The story of Insulin

Today, I'm relaying the story of how Insulin was discovered and ultimately led to the saving of countless lives.  Honestly, this whole scientific tale has been rather neglected and not given the attention it deserves. 

Insulin is a hormone that in short regulates blood sugar levels, to allow the body to use energy efficiency. So, if you eat a sugary meal, your blood sugar levels would increase- but the body needs to get this sugar out of the blood and to the cells that can actually use it .If the sugar remains in the blood, blood sugar levels get too high and this results in quite a bit of damage to blood vessels and nerves, along with kidney damage and eye damage. 

For those with type 1 diabetes, or diabetes mellitus, Insulin is not produced effectively.  This is due to the immune system attacking the beta cells in the pancreas. The beta cells are located in clumps of cells called the Islet of Langerhans and are where Insulin is secreted from.  Type 1 diabetes is ultimately an autoimmune disorder and if the beta cells are damaged, insulin cannot be secreted. 

Type 1 diabetes was once known as the sugar sickness, and it wasn't until the discovery of Insulin that it could actually be treated effectively.  Ultimately, it was a death sentence- and a cruel one.  A person's blood sugar would be dangerously high but there would be no way for the body to actually use this sugar to generate the energy it needed to keep going. 

As a result, other sources of energy would need to be press-ganged into service. The body would start frantically breaking down fat and muscle to get energy. But this would result in a condition called Diabetic Ketoacidosis (DKA) where the blood would become too acidic. Combine this with high blood sugar and you end with up life threatening medical emergencies - meaning that before the 1920s, many of those with type 1 diabetes could not hope to live for long. 

Until Insulin was discovered, one of the only effective treatments would be the starvation diet. This is pretty much as awful as it sounds, and it did not hope to actually cure diabetes. It only hoped to prolong life by severely restricting food intake. This was a desperate attempt to lower blood sugar with patients often consuming only a few hundred calories per day. This of course made already ill patients thin and fragile.  

With this background established, the story of Insulin can begin proper. The story of Insulin actually begins in 1869, more than 50 years before Insulin itself is identified. In Berlin, a medical student by the name of Paul Langerhans (is this name ringing a bell to anyone yet?) looks down a microscope at a pancreas. At this stage, the pancreas was known to have some role in digestion - but not much else. Paul Langerhans noticed some tissue clumps scattered throughout the pancreas which he referred to as 'little heaps of cells'.  As to what they did... well, that wasn't yet clear- but stay tuned as their importance will soon be discovered. 

Paul Langerhans. 

20 years later, the story takes us over to the University of Strasbourg where we encounter Oscar Minkowsi, Joseph von Mering, and several dogs. After removing the pancreas from a healthy dog, these physicians discovered sugar in the dog's urine. This allowed for a link between the pancreas and diabetes to be established for the first time. Sadly, the dog ultimately would have died from complications of the pancreas being removed. Before I go any further, I should now mention that experiments on dogs play a very important role in this story.  In the late 19th century and early 20th century, there were no formal animal welfare regulations. It is undeniably sad that these dogs died and modern standards would result in these experiments being subject to strict ethical oversight. However, in the context of the time, these experiments paved the way for insulin and ultimately saved millions of lives. In short, the sacrifice was not in vain and was in fact life-transforming. 

Oskar Minikowsi (left) and Joseph von Mering (right). 

In 1893, it was then suggested by Edouard Laguesse in 1893 that the little heaps of cells discovered by Paul Langerhans may play a regulatory role in digestion. He named them as Islets of Langerhans in honour of their discoverer. 

Edouard Laguesse. 

Now the story takes away from Europe, and we head over to Virginia, USA.  In 1903, medical student Eugene Lindsay Opie notices morphological changes in the Islets of Langerhans in patients with diabetes. He concludes that Diabetes occurs when the Islets of Langerhans are partly or wholly destroyed. 

Eugene Linsay Opie, 1903.

The story then lags a little bit with many researchers attempting to isolate the secretions of the Islet of Langerhans. George Ludwig Zuelzer had some success in 1906 when he injected pancreatic extracts into diabetic dogs. He also created an extract called 'Acomatol' which was derived from calf pancreases and injected into it a comatose dying diabetic patient. The patient showed some improvement but suffered from side effects and died once the supply of Acomatol was exhausted. 

George Ludwig Zuelzer. 

In 1911 and 1912, Ernest Lyman Scott was working at the University of Chicago, as a master's student under the supervision of Anton Carlson.  It's not quite clear what his exact role in this story was but it is known that he worked independently on dogs with disrupted pancreases to isolate an 'active principle' that had anti-diabetic effects.  His 1911 master's thesis suggests that he isolated a protein that had clinical benefits in diabetic dogs.  This protein was likely Insulin. However, this version of the thesis was not published until 1966. Instead, Anton Carlson published an edited version in 1912 in Scott's name.  This version was arguably more cautious with softened claims.  Carlson was sceptical that Scott had actually isolated the 'active principle’ and it is also important to note that Scott's extracts were impure and not adequate for clinical use.

Ernest Lyman Scott.

In 1914, Zuelzer was still working to seek a cure for diabetes. With the help of the company Hoffmann-La Roche, his pancreas extracts reached a higher level of purification and injected into diabetic dogs. Sadly, the dogs developed convulsions and unfortunately died.  Zuelzer believed this was due to the preparation not being sufficiently purified. In actuality, it was most likely caused by an overdose of insulin causing hypoglycaemia.  

At Rockefeller University in 1915 Israel Kleiner discovered similar results to Scott and demonstrated how pancreatic extracts caused hypoglycaemia -low blood sugar. Crucially, Kleiner's work used blood glucose measurements, rather than urine sugar which many of the other researchers had focused on. In a healthy human body, glucose is not found in the urine due to the kidney reclaiming it, but in diabetes, excess glucose spills into the urine as the kidney is unable to cope with it all. 

Israel Kleiner, 1915

In 1916, Nicolae Paulescu made significant strides. He was successful in significantly normalising blood sugar levels in dogs by injecting a pancreatic extract. 

Nicolae Paulescu, 1897

Unfortunately, the work of Zuelzer, Kleiner and Paulescu was significantly disrupted by WW1. Kleiner was not able to return to his work, and in Germany, Zuelzer's lab was turned over to the German military. Paulescu also had to interrupt his experiments as he was called into service in the Romanian army -although later returned to this story...

The story doesn't pick up again until 1920 in Canada. For those who read my article last week, you might recall the names Frederick Banting, Charles Best, John Macleod and James Collip.

Frederick Banting. 

In spring 1921, Frederick Banting is travelling to Toronto to speak to John Macleod at the University of Toronto. Banting was able to conclude in October 2020 that the islet secretion couldn't be effectively extracted as digestive secretions were breaking it down. Banting realised that blocking the pancreatic duct would lead most of the pancreas to die - but the islets would remain intact. He then reasoned that a pure extract could then be extracted. He had an idea: if the pancreatic ducts of a dog were ligated, and the dog kept alive until the pancreas degraded, a pure extract could then be obtained from the Islets. This is what he travelled to Toronto to speak to Macleod about. 

John Macleod

Initially sceptical, Macleod agreed to provide lab space. He also arranged for two undergrads to assist Banting; Charles Best and Clark Noble. As Banting only required one assistant, the two flipped a coin to decide who got to be Banting's lab assistant. Charles Best won and was kept by Banting for the whole summer. In July 1921, the two were able to extract a secretion from islets of a dog with a degraded pancreas and injected it into a diabetic dog. This extract reduced the blood sugar of the diabetic dog by 40% in an hour.  In Autumn 1921, Macleod moved Banting and Best to a better laboratory began paying Banting a salary from his research grants and helped them publish their results from the next set of experiments. As the process of duct-tying dogs and waiting several weeks became time consuming, they started using the fetal calf pancreases - as these had not yet developed digestive glands. By December 1921, Insulin had also been extracted from the adult cow pancreas. The main task now was to purify the secretions. Macleod discounted all other research to concentrate on this and invited James Collip to help with the purifications. 

Charles Best and Clark Noble, c.1920

James Collip, c. 1930

Meanwhile, Paulescu returned to his experiments and published four papers about his work. He was able to conclude that pancreatic extracts that he called pancrein, reduced blood sugar and urinary sugar, and that the pancreas secretes an internal hormone responsible for regulating carbohydrate metabolism.  

This was different to the work of Scott, Zuelzer and Kleiner as his experimental results were much more consistent- and complete He had conducted a series of controlled experiments, published detailed results and was the first to conclude that the pancreas produces an anti-diabetic hormone. 

Anyway, back in Toronto... 

Things started to move at a rather breakneck speed from there and on the 11th of January 1922, less than a year after Banting's journey to the University of Toronto, the story takes us to Toronto General Hospital, at the bedside of a dying 14-year old diabetic.  

Leonard Thompson, c.1930

Leonard Thompson received the first ever injection of insulin. However, the extract was impure, and Thompson had a severe allergic reaction.  Over the next 12 days, Collip worked to improve the extract, and Leonard Thompson received the second ever injection of insulin on the 23rd of January.  This time no side effects were seen. 

Things then moved even faster from there. In August 1922, Elizabeth Hughes, the daughter of American statesman Charles Evan Hughes, received the third ever injection of insulin from Dr Banting in Toronto. She became the first American to receive Insulin. At the time of her injection, the 15 year old Hughes weighed 20kg and had been surviving on a starvation diet of less than 800 calories per day. After receiving Insulin, she recovered quickly and within two weeks was placed on a 2200-2400 calorie weight gain diet. At the time of her death in 1981, she had received approximately 42,000 insulin injection.

Elizabeth Hughes, c. 1930

Also in 1922, twenty-two year old James D Havens become the first person in America to receive insulin.  He received his insulin from John Ralston Williams who imported it from Toronto to Rochester.  Havens died at the age of 60 from cancer. 

James D Havens with one of his children

Tensions began to rise between the four co--discovers to the extent that Collip threatened to separately patent his purification process. Meanwhile, Macleod and Banting were reluctant to patent their process on grounds of ethics - but concerns began to rise that a third party could hijack and monopolize the research, and that safe distribution would be difficult to guarantee. In the end, the four co-discovers along with John G. FitzGerald, the director of the public health institution Connaught Laboratories wrote to the University of Toronto to propose an arrangement. This arrangement ensured that the production of Insulin would be licensed to reputable pharmaceutical companies, with strict quality controls- but no one including the discovers could benefit financially. In short, no one could impose monopolistic pricing. The discovers symbolically sold their patent to the University of Toronto for 1 dollar in January 1923.

John G. FitzGerald.

By late 1923, large scale commercial insulin derived from cow and pig pancreases became available in the United States and Canada. Honestly, it is insane how fast it all moved. The speed at which the drug was purified and entered the public domain following its discovery was extraordinary. Ironically, despite advancements in scientific technology making protein purification faster and more precise, it would now take so much longer to actually get lab results into clinical practice. This isn't due to slower science or reduced research but a reflection of modern safety and ethical standards. 

In 1923, The Nobel Prize Committee awarded the prize to Frederick Banting and John Macleod.  As explained in my article last week, it is still subject to debate why Paulescu didn't receive the Nobel Prize. He wrote to the Nobel Prize committee claimed he had discovered Insulin first. Zuelzer also wrote to the Novel Prize Committee claiming he had done it first. Some would argue that Scott also deserved to be acknowledged as did Zuelzer.   Ian Murray, a professor of physiology in Glasgow, vice-president of the British Association of Diabetes and a founding member of the International Diabetes Federation claimed in 1971 that Paulescu did not receive the recognition he deserved. A potential reason for this is Paulescu being a victim of bad luck.  Banting and Best did reference Paulescu in their 1922 paper - but made a mistranslation.  Paulescu's papers were written in French and neither Banting nor Best were particularly fluent. They claimed that Paulescu had stated injections of pancreatic activity does not have any effect on blood glucose. Paulescu had actually stated the complete opposite. This mistranslation even became part of the written evidence supporting the Toronto group's claim of discovering insulin.  (My thanks to Thomas Annesley, Professor of Clinical Chemistry at the University of Michigan Medical School for teaching me this). 

 The work in Insulin continued, with the next task being to determine its structure and what it actually was.

Michael Somogyi, Edward A Doisy and Philip A Shaffer provided evidence that Insulin was a protein in 1924. This was proven when Hans Jensen and Earl A Evans Jr isolated amino acids phenylalanine and proline in 1935. In 1955, Frederick Sanger characterised the amino acid structure of Insulin-he was awarded the 1958 Nobel Prize in Chemistry for this discovery. This allowed for synthetic insulin to be produced for the first time at the lab of Paynayotis Kasoyannis (University of Pittsburgh) and Helmut Zahn (RWTH Aachen University) in the 1960s.  In 1969, Dorothy Hodgkin used X-ray crystallography to determine the entire 3-dimensional structure of insulin. She had previously received the Nobel Prize in Chemistry in 1964 for her work in crystallography. 

Hans E Weber was able to discover a precursor to Insulin in 1974. This became an important molecule to study transcription and translation. 

In 1978, another major breakthrough was made by Arthur Riggs, Keiichi Itakura and Herbert Boyer when a genetically engineered synthetic human insulin was successfully produced using E. coli. This biosynthetic human insulin became commercially available in 1982 under the brand Humulin.  Most insulin now produced worldwide is biosynthetic human insulin. Whilst animal-derived insulin was effective and many lived long healthy lives whilst taking it, it had slight differences in structure which could result in immune reactions, some injection-site reactions and some insulin resistance. The biosynthetic insulin reduced in less immune reactions and was also purer.   

This now brings the story up to now. However, the story is still not finished. Research is still ongoing to improve many aspects of Insulin production and delivery.  Insulin Degludec, under the brand name Tresiba, is a long acting insulin designed to act over 42 hours, reducing the number of injections required. Delivery methods are also being researched along with 'Smart Insulin' - an insulin that would only activate when blood glucose. Some research focuses on creating a pancreas using stem cell-derived islet of Langerhans and using CRISPR to deliver the insulin gene allowing the body to produce insulin itself.

Some of these developments may seem a bit outlandish and some - like a stem cell pancreas are still potentially years and years away. But once, it might have seemed outlandish that a cure to diabetes would even exist.  So many researchers played significant roles in this discovery. Many of us will know someone with type 1 diabetes or maybe are diabetic themselves. This life-saving hormone is easily taken for granted but it is built on decades of experimentation, persistence and sacrifice. The work of these researchers transformed a fatal disease into a manageable condition. This article and remembering their contributions is more than a historical exercise- it's also a way to honour the lives saved and the impact this discovery still plays. 

 

Scientists who maybe should lose or share their Nobel Prizes

 

The Nobel Prize is an extremely prestigious award. For scientists, there are three categories up for grabs - Chemistry, Physics and Physiology or Medicine.  Whilst the majority of recipients are widely regarded to have deserved and were rightfully given their prizes, there are a few cases where the decision of the Nobel Prize Committee had been criticised and has some persisting controversy.

Here are, in no particular order, some examples of these cases. Some of them are more well known than others but they all represent cases where a wince and a comment of 'oh dear!' may characterise the decision of the Nobel Prize Committee that year: 

1.Antonio Caetano de Abreu Freire Egas Moniz:

Antonio Egas Moniz

Egas Moniz, alongside Walter Hess, won the Nobel Prize in 1949 for his discovery of the therapeutic value of leucotomy. Otherwise popularly known as lobotomy- although this term was not used until other physicians -notably Walter Jackson Freeman II and James W. Watts developed a modified technique.

Lobotomy for those that don't already know is neurosurgical procedure that was designed to treat mental illness by severing nerve pathways in the prefrontal cortex. This region of the brain is responsible for processing and adapting thinking to meet goals but also controls speech formation, gaze, memory and risk processing. The general idea was to induce a calm docile state by severing what Moniz thought were abnormal neural connections. He also believed that removing white matter fibres from the frontal lobe would improve mental illness. He later developed a technique with his staff member Pedro Almeida Lima that involved the leucotome - a needle-like instrument with a wire loop that allowed them to separate white matter fibres. To Moniz's credit, he never performed a surgery himself. 

A version of the leucotome. 

Lobotomy would often result in patients becoming incontinent and epileptic, and experiencing severe changes in personality and ability to function independently. Many also suffered a reduced consciousness, dementia and in some cases death. In the best cases, patients would be left with inertia, a lack of response, self-awareness and emotional numbness. 

Before being awarded the Nobel, Moniz was already being criticized for his understatements of complications, his lack of documentation and for failing to following up with patients. Moniz defended himself by claiming that the procedure was always safe but did concede that patients did not benefit if they had already deteriorated from the mental illness. He also stated that the behaviour and personality deterioration that could occur was outweighed by the benefits- i.e. the debilitating effects of the illness would be reduced. 

It is important to note that leukotomy was a desperate attempt to gain some control over mental illness. There were very little options for the treatment of severe mental illness.  However, even before Moniz won the prize, medical associations such as the AMA, and psychiatrists were denouncing the procedure and warning against it. The damage was visible. To make it even worse, Moniz's poor scientific practice made his subsequent awarding of the prize even more incredulous and bewildering.

 

2.Johannes Andreas Grib Fibiger. 

Johannes Fibiger

Fibiger was awarded the Nobel Prize in Physiology or Medicine in 1926 for discovering a new kind of roundworm that he claimed caused stomach cancer. Unfortunately for him, he had made some rather significant errors in his data analysis.

In 1907, Fibiger was investigating tuberculosis in lab rats and found some stomach tumours in rats collected from Estonia.  He then discovered that these rats also had nematodes (worms) with eggs. After investigating for years, he identified the nematode as a new species and demonstrated that this nematode could induce stomach cancer.  This conclusion was supported by two Japanese scientists Katsusaburo Yamagiwa and Koichi Ichikawa who proved that carcinoma could be produced in rabbits by painting coal tar on their ears. Whilst this could now regarded as unethical, in those days, virtually nothing was known about cancer development. Yamagiwa was nominated for the Nobel Prize alongside Fibiger but was excluded in favour of Fibiger. 

Koichi Ichikawa (left) and Katsusaburo Yamagiwa (right)

Whilst Fibiger's discovery of the stomach cancer-causing worm went mostly unchallenged in his lifetime, questions were raised after his death.  Vitamin A deficiency was found to cause tumours and cancers, and Fibiger's rats had been fed a vitamin A -free diet. Meanwhile, it was also found that the cancerous tumours that Fibiger had found were not cancerous at all. It was then concluded that the worms had caused tissue irritation. Combined with the vitamin A deficiency, this caused non-cancerous tumours to develop.  Fibiger unfortunately was very wrong. 

To be fair to him, Fibiger never, as far as can be proven, set out to defraud anyone. However, his awarding of the Nobel prize - and the exclusion of Yamagiwa, is considered by some to be a mistake and undeserved. 

 Still, it may be interesting to know that whilst Fibiger's worm G. neoplasticum, does not cause cancer, some species of flukes- another type of worm have now been linked to cancer development in humans.

 

3. Frederick Banting and John Macleod

Frederick Banting (left) and John Macleod (right)

In 1923, Frederick Banting and John Macleod were awarded the Nobel Prize in Medicine for the discovery and extraction of Insulin.  In 1921, Banting - a surgeon by training, travelled to Toronto to explain an idea he had to John Macleod.  Earlier research had discovered that an extract from a certain region of the pancreas had a normalising effect on blood sugars.  Banting believed that this region could be isolated and would secrete a substance that could treat diabetes. Macleod was sceptical but agreed to giving Banting some lab space - and an undergraduate to assist him; Charles Best.

                                    

                                          Charles Best. 

 Together, Banting, Best and Macleod were able to extract insulin from a pancreas. Macleod invited biochemist James Collip to join their efforts to purify insulin, and in January 1922, insulin was injected into a diabetic for the first time- 14 year old Leonard Thompson, who was dying at the Toronto General Hospital. Before this discovery, type 1 diabetes were almost uniformly fatal.  Leonard Thompson did die 12 years later- but of pneumonia. By November 1922, Insulin was offered for sale to the general public, saving countless lives.  Notably, the patent for Insulin was sold to the University of Toronto for 1 dollar. 

James Collip

In a move that is known to have incensed Banting, Best was not included or mentioned in the awarding of the 1923 Nobel Prize. Neither was Collip. Banting shared his prize money with Best, whilst Macleod shared his with Collip.  In addition, it has also been argued that Nicolae Paulescu should also have been awarded the prize. Whilst these four were the first to use insulin on human patients, Paulescu was the first to actually determine that the pancreas was producing a substance that could normalise blood sugar levels. He had developed an extract that he injected into a diabetic dog - treating its diabetes.  

Ultimately, three people that arguably deserved to be acknowledged as well were excluded and did not receive recognition for their efforts. The Nobel Prize cannot be shared more than three ways so at least two people would have been left out. So, should the Nobel Prize have also been awarded to the man who started it all off, the man who was instrumental to its extraction for human use, or the man who was essential to its purification? This three-person limit is still widely criticised and is still controversial- modern science often requires on massive collaborative teams.

Below are some key examples where the awarding of the Nobel Prize excluded key contributors and are still widely debated:

1.Otto Hahn. 

  

Otto Hahn 

He was awarded the Nobel Prize in Chemistry in 1944 for the discovery of nuclear fission. However, whilst Hahn performed the experiments, Lise Meitner provided the theoretical explanation and coined the term 'fission', alongside her nephew Otto Frisch. Fritz Strassmann was also essential to the work and was a full collaborator. All three were essential for the discovery of nuclear fission.

Fritz Strassman and Lise Meitner

2. Antony Hewish and Martin Ryle.

 

Antony Hewish (top) and Martin Ryle (bottom).

Antony Hewish and Martin Ryle were awarded for the Nobel Prize in Physics in 1974 for the discovery of pulsars - or rotating neutron stars that emit radiation. This excluded Jocelyn Bell Burnell, the graduate student working under Hewish.  Bell Burnell was the one to actually see a strange signal and spent three months ruling out errors. Some nights, she would be reviewing at much as 96 feet of paper data. Initially, Hewish insisted the signal was caused by interference in the detection equipment and was not significant, forcing Bell Burnell to be persistent in the face of scepticism.  Bell later opined that being a student and a woman 'demoted [her] standing in terms of receiving a Nobel Prize'. 

Jocelyn Bell Burnell.

3. James Watson, Francis Crick and Maurice Wilkins.

I've already moaned about this in my articles about photograph 51 and Rosalind Franklin so I'll rein it in. But the structure of DNA would not have been made without Rosalind Franklin, and she should have received a prize - even posthumously. 

I'm going to call it a day there before I end up having another rant/ vent about the lack of justice done to Rosalind Franklin and how James Watson was an absolute ar*e. 

Overall, these are just some notable examples where the Nobel Prize may not have gone to the person -or the people that deserved it. There are likely other cases where scientists were excluded, or the research arguably didn't call for the awarding of such a prestigious prize. Some would argue that the Nobel Prize itself and its rules about who can receive an award just isn't suited for modern science anymore.  However, the Nobel Prize is still, quite rightly, regarded as an honour. In a world where science isn't always given the respect and recognition it deserves, it can be argued that any awards that give recognition and acknowledge to its advancement should be embraced, no matter what mistakes might have once been made. 

 

Honest Protocols: Antibodies, visualising, and stripping

 We finally made to the end of the western blot honest protocols series!  

At this stage, our membrane has been left to block in the cold room overnight and now we are going to start working towards the visualisation stage - where we will find out two things:  the protein you wanted in the first place was in the lysate, or the western blot didn't work.

So, to see if the protein is present, we are going to use antibodies. Antibodies are the reason your western blot looks like it does and produces those bands. Each of those bands is where an antibody has bound to your target protein. 

There’re two types: the primary and the secondary.

The point of the primary is to bind to the target protein - or the protein that you want to know is within your lysate. Most of the primary antibodies are monoclonal -meaning they will only bind to one protein (in theory) meaning that you need different primary antibodies for every protein you want to detect. 

The secondary binds to the primary antibody and is there to amplify the connection between the primary and the protein of interest. Whilst the primary antibody finds the protein, the secondary antibody makes it visible. The secondary antibody isn't specific, and like the primary antibody is usually produced in a rabbit or goat. Weird but there we go. If you use a goat primary antibody, you also have to use a goat secondary. Same goes for rabbit. 

Handy diagram on how primary and secondary

antibodies work. 

So, what you need to do is apply the primary antibody to the membrane, leave it for a while and then add the secondary antibody. In between this, you want to be washing off the excess antibody to stop any background noise. You also want to be washing off the blocking solution. 

So: 

1) Pour away the blocking solution and leave your membrane floating in PBS-T* on the shaker in the lab (not the one in the cold room). 

*PBS-T is phosphate buffered saline with a small amount of Tween-20, a detergent added. It keeps the pH stable, and the Tween helps prevent the antibody from binding to anything it shouldn't. You can also use TBS- which is Tris buffered saline. Both work more or less the same.  

2) Leave it for five minutes.

3) Pour off the PBS-T and add more PBS-T. 

4) Repeat steps 2-3 about three times. Lose track of how many times you have actually done it. 

5) Check to see whether you still have some antibody solution left over. Much to your disappointment but not to your pressure, you don't. 

 6) Sigh when you realise that this means you need to make 5% BSA-PBS-T solution.

   *BSA-PBS-T is BSA mixed with PBS-T.  Typically, it's about 2.5g with 50ml of PBS-T. The best way to do it is weigh out 2.5g of BSA, add it to a tube and then top it up into 50ml.

7) Pause briefly to wipe up the BSA you split all over the balance.

8) Spend the next 10 minutes frantically shaking the tube up and down in an attempt to dissolve the BSA. 

9)Have a rare moment of forward thinking and remember that you may need more of the 5% BSA-PBS-T. So, make up another tube and stuff in the fridge for later.

10) Retrieve your vial of primary antibody from the -20 freezer. 

11) Start thinking about what concentration of antibody you will actually need- and how much of the antibody solution you will need. Decide on 15 ml of BSA-PBS-T and add 15 ul of antibody*

  *Typically, you use a concentration of 1:1000, for the primary antibody and see what happens. You can try and increase the concentration later. Some antibodies work with a lower concentration, or some need a stronger. concentration. There's no way to know until you try it. 

12) Pour away the PBS-T on your membrane and add about 10 ml of the antibody solution onto the membrane. Like with blocking, check the membrane is moving. 

13) Incubate with primary antibody overnight on shaker in cold room*

 *Or one hour on the shaker at room temperature. Depends on how lazy you are being. But it has to be at least one hour if doing it at room temperature.

14) Wash the membrane with PBS-T five times. 

 15) Retrieve your secondary antibody from the 4 degrees freezer and make up your secondary antibody solution* with 5% BSA-PBS-T.

      *The secondary antibodies are stronger so use a concentration of about 1:3000. Which means about 5ul for 15 ml. And remember to check whether your primary was a goat or a rabbit. Your secondary has to be the same. 

16) Remove antibody from membrane*. 

   * Just pour it back into a conveniently labelled test tube (for the love of God, make sure the test tube is labelled). The antibody can be re-used but no more than 5 times at the absolute most. 

17) Pour about 10 ml of secondary antibody on to the membrane and leave it to it for 1 hr on shaker at room temp*.

* No matter how lazy you are feeling, the secondary cannot be left on overnight. It causes background noise.

18) Wash three times in PBS-T, like you did with the primary.

19) Take a deep breath and brace yourself for your fate. After all this work and hassle, you get the fun of visualising it and seeing if you all your hard work was for nothing or not.

20) Sadly wander over to the visualiser to book a slot. 

21) Unfortunately (or perhaps fortunately), it's not available right now.

22) Book a slot for about an hour from now and leave your membrane sulking/soaking in PBS-T*,

*You could leave the membrane soaking in PBS-T for a week and it would be fine (That is not scientific advice).

23) Come back after an hour and grab your ECL from the cold room.

   *ECL is the detection reagent you will use to see your protein bands. Your secondary antibody usually has something called HRP attached to it. This reacts with the ECL and gives off a small amount of light. It's not visible to the human eye- so you need the gel visualiser to see it. This light is what forms a band. Which means in theory, you only get the bands where the proteins are.  You can then use the ladder to determine how big the bands are. This is how you can confirm that the band is the protein you actually wanted. 

How ECL works on the primary antibody and 

secondary antibody. 

24) Just before* you are about to use it, make it up using a 1:1 ratio of reagent A to B. 

*ECL degrades quickly so you don't want it sitting around for long. Depends on the membranes but I usually made it up with 5ml reagent 5 A and 5ml reagent B. I'm not making that up - they are actually called A and B. 

25) Forget all you know about ratios.

26) Eventually figure it out. 

27) Nervously approach the visualiser. Tentatively turn it on and try to remember how to use it*.

*Every visualiser is different but there's usually instructions nearby. 

28)ECL didn't show anything, so you try with ECL plus*

   *ECL Plus is basically a more sensitive version of ECL. Sometimes, a protein might be on the membrane but at a really low amount so you need to use something more sensitive.  For ECL plus, you use 1:40 ratio of reagent A to B. 

29)That also doesn't work so you decide to try West Femto.

*West Femto: Even more sensitive. If you are at this point, there's not that much hope but there is still a possibility!!  1:1 ratio of reagent A to B

30) Pray. Pray. Pray.

31) Pray again to the western blot gods. They do exist and they are watching over you.

32) Let's pretend this hypothetical western worked and you actually got the bands you wanted. Happy days! 

What a successful Western should look like.

Either side is the ladder whilst the thicker black band

 about a third down is the protein of interest. Some background bands 

can also be seen but they aren't getting in the way.

33) Soak membrane in PBS-T for three times, 5 minutes each. 

34) Find the stripping buffer*

* You want to remove the secondary antibodies from the membrane but keep the actual proteins in your sample present. This means you can use a different antibody on the membrane to see if another protein is present. What you usually do is use GAPDH. This is your control antibody. And you use it to basically prove that everything has gone right. Because everything has GAPDH. GAPDH is strong so a concentration of 1:5000 is usually okay.  Stripping buffer is usually pre made and is located somewhere in the lab. Good places to find it are above the shaker or on the bench of the person who spends their entire life doing westerns.

35) Remove the PBS-T and add 5ml of stripping buffer.

36) Leave for 5 minutes at room temp on shaker. 

37) Wash again with PBS-T, 5 minutes each.  

38) Repeat steps 13 to 33.

39) Celebrate when you see your GAPDH bands.

40)Discard/ keep your membrane. Your choice. If keeping, leave it to dry, wrap it securely in clingfilm and put it in the -20 freezer. If discarding... well, just throw it in a bin. 

At best, it's been about three days since you started this. At worse, it's closer to a week. But congrats, you made it through your first western!  The first of many. I would like to say that you will start to enjoy it....  but you might find yourself resigned to it and stop hating western blots as much as you do at first. At least it feels absolutely amazing when it works. There is no feeling quite like it. 

 

Honest Protocols: Transferring and blocking

 

Honest protocol: transferring and blocking!

This is my latest post in my honest protocols series. At this point, we have finished with the electrophoresis and can now move onto the biggest hell that any scientist will ever have to face... the western blot!

 The point of the western blot is to see what antibodies are able to bind to the proteins that have separated out on the gel. I'll explain a bit more about what the antibodies do and how this helps us identify what proteins are present next time, but what we need to do is get those proteins from the gel onto the western blot membrane. This membrane is a thin porous sheet that immobilise the proteins. Once the proteins are on the membrane, you can start using antibodies to see what proteins you actually had in the lysate in the first place.  The real problem with westerns blots is that you can't see if it worked or didn't work until the last possible moment.  At this point, the electricity for your electrophoresis should have been switched off. The gel can sit in the tank quite happily whilst you get the next stage sorted. 

1) First, decide what membrane you actually want to use*. If using PVDF, soak it in methanol for 5 minutes. Grab the methanol from the hazard cupboard, start to pour it out.... then realise that you should really be doing this in the chemical fume hood. Stop yourself before gassing out the entire lab and sheepishly shuffle to the fume hood. 

*Sometimes, you don't actually get a choice. Your lab stocks one kind and that's that. There’re two types of membrane: nitrocellulose and PVDF membranes. Both have their advantages. Nitrocellulose is better for small proteins but insanely fragile. PVDF is better for your high molecular weight proteins and is also less fragile. You have to soak it in methanol before using it because it is hydrophobic and won't work otherwise. Nitrocellulose can be used instantly. 

2) Retrieve the stacks* from the random-looking cupboard they are usually left in, after spending about 10 minutes trying to work out where they are. 

*Stacks are basically a 'stack' of filter paper. It's designed to be thick. Some labs will just buy thick filter paper, others will buy standard filter paper, and you use stack layers of it together to make a 'stack'. There's always some floating around. If you're lucky, some kind and organised person will have cut it into nice convenient sizes.  Other times, you spend another 10 minutes trying to hunt down thick enough scissors. 

Literally just thick filter paper. 

3) Hunt down your pre-made transfer buffer* that you made in a rare display of forward thinking.

*This is the conductive solution that moves the separated proteins from the gel onto the membrane.  It's a buffer agent - usually Tris, to maintain the pH combined with some methanol to get the protein transferring to the membrane and a little bit of SDS which helps move your large proteins. Sometimes it arrives pre-made which in theory makes your life easier. 

4) Discover that someone has used your transfer buffer. Be angry for a few minutes and then remember that your lab bench neighbour asked if she could and you were generous enough to say yes.

5) Regret your generosity for five minutes.

6) See that she has made some up. Pop your head around the cold-room door, see that she is in the lab, and yell out across the lab to get her attention. 

7) Ask if you can use her buffer. Receive answer in the affirmative*.

*If the answer is in the negative, odds are there is another helpful person who will let you use theirs. Or will help you make some of your own up. 

8) Pour transfer buffer over your stacks and leave them to it whilst you hunt down a transfer cassette*

*The transfer cassette is what you seal your gel, membrane and the stacks in. It stops the gel and membrane from moving about, keeps them in constant tight constant and applies a constant electric field that pushes the proteins onto the membranes.  

A transfer cassette with the lid on the left. 

9) Start assembling the transfer sandwich *. Lay a stack down on the cassette and place the membrane on top.  DO NOT TOUCH THE MEMBRANE*.

*This is actually what it is called. 

*Otherwise, the protein on your fingertips gets transferred too and you end up with a massive fingerprint. 

10) Hunt down a roller* and run it over your transfer half- sandwich.

Roller. 

*It looks like a mini version of those rollers you use in painting walls. The point of it is to get rid of any air bubbles. They get in the way of the electric field and stop the protein transferring. 

What happens when you don't roll.. 

11) Fish out gel and it's plastic casing from its tank. Decide that you only used this buffer once and you can't be bothered to make up another load for your next western blot* Pour it back into its bottle, only spilling a quarter of it in the process.

*You can use a running buffer two or three times. No more than that. Some don't bother and just make a new one. 

12) Crack open the gel from its plastic casing using the handy cracking tool that some kind lab member lets everyone use and the arrows on the plastic surrounding the gel. Pray to any god you do or do not believe in that the gel will not break in the process. 

Handy cracking tool. It probably has a proper name. 

13) Somehow place the gel neatly on top of the membrane *.

*Easier said than done. The way I used to do it was use the cracking tool to take off half of the plastic casing. The gel should be lying on top of the other half. From there, you have two options. You can gently lift it up by its corners using plastic spatulas and lay it on the membrane. Or, you can flip the plastic casing over so that the gel is facing the membrane and use the spatula to slowly nudge it off the plastic and onto the membrane bit by bit. At some point in your western blot life, the gel WILL break and you will cry. But you might still get some data.  Put the broken pieces on the membrane and try to piece it together.  This is also why we used the ladder to denote each side of the gel.  If you flipped the gel + plastic, or the gel at any point, it can be hard to work out what well actually had what sample. You put sample 1 in well 1 but is well 1 now on the left or the right? If you did two ladders on the left hand side, you know well 1 is always the one next to the two ladders. 

The plastic spatula things- 

these are actually known as gel releasers. 

14) Roll it out again and place another stack on top of the gel*

*The reason you do it this way with the gel on top of the membrane is because the electrical field that the transfer cassette is creating acts downwards. The top of the transfer cassette will be negative whilst the bottom of the cassette will be positive.  Proteins are negative so they will move away from anything negative but move towards anything positive. 

Handy diagram of the transfer sandwich.

15) Pour some transfer buffer on top.

16) In one swift clean motion, slam* the top of the transfer cassette on top of the sandwich and lock it in place by turning the dial. 

   *Okay, you don't actually have to slam it. But you don't want the lid of the transfer buffer to dislodge any part of the sandwich and add air bubbles. You can't just slide it in place or shuffle it about. It has to be placed in exactly the right location in one motion.  

17) Blindly panic that you did the transfer sandwich wrong as you can't remember what you actually did.

18) Wonder if you should open it up and check... but you also don't want to have to reassemble and get the roller out again as you are also scared that you will break the gel.

19) Shove the cassette in the machine. Wonder what voltage you should use and for long*.

*It depends on the size of the protein.  You want the proteins to move off the gel and onto the membrane but if you leave it too long, the proteins can move though the membranes and into the stacks which you don't want. Large proteins need longer time and potentially more voltage Honestly at first, it's a bit of trial and error and see what happens.

Transfer machine with two cassettes.

20) Decide on 10 minutes and 25 V*.

*This is a standard reasonable time. Some transfer machines don't give you the option of choosing voltage but say things like 'moderate weight', 'large weight' and 'mixed weight'. Problem is, they don't say what counts as 'large weight'. 

21) After 10 minutes, use tweezers to lift up the corner of the gel. If the ladder has moved off the gel and onto the membrane*, it's done. 

*You can see the ladder on the gel when the electrophoresis is done before of the dyes in it. When the transfer is done, there should be nothing left of the ladder on the gel, and you should instead see it on the membrane. 

22) Cry with absolute relief. You didn't want to slot it back in the machine for two minutes intervals and check in on it each time as it's flipping boring and irritating. You also think you moved the gel when checking on it*.

   *If you move the gel before the transfer is done and then put it back, unless you put it back in the EXACT same spot, you end up with double proteins transfer -two overlapping protein bands when you should only have one band.

Double protein transfer. 

23) Dissemble your transfer sandwich. Start to throw away your stacks then remember that your lab is supposed to be eco-friendly and you can re-use them.  Deposit them in some plastic tray somewhere on your bench to dry off. Discard the gel * after squeezing it and playing with it for a few seconds, enjoying the sensation. 

*After all that work, it’s now useless to you.

You membrane should look a little like this at this stage.

All you can see is the ladder and none of the protein 

from the lysates. 

24) Decide that you are too lazy to continue with this and decide to block * the membrane overnight.

*Blocking is what you do to your membrane to make sure only the proteins in your lysates actually bind to the antibodies you will apply later.  It's basically covering any part of the membrane that doesn't have protein on it with unreactive proteins. They won't bind to any antibodies.  You can either do it for one or two hours at room temperature or just leave it overnight in the cold room.  Like all stages of the western blot, you will not know if it worked until the absolute last moment. 

25) Hunt down your blocking buffer * from the fridge. 

*You're in a fancy lab so you are using Bovine Serum Albumin. Milk also works so some labs just have milk powder which you make up with distilled water.

26) Grab your plastic western blot membrane holder thing*

*Any plastic thing with a lid works well. There's probably some lab supply company selling official western blot blocker holders, but you can use almost anything. 

It's like an old video cassette. Think they are officially 

called 'blot trays'. But anything with a lid works. 

27) Pour the blocking buffer all over your membrane in the plastic holder thing.

28) Wander over with the plastic holder + membrane + buffer over to the cold room and find some room on the shaker *.

*The shaker keeps the membrane moving at a steady speed. It's making sure that the blocking buffer is going to reach every part of the membrane evenly.  

Like this. 

29) Check that the membrane is moving independently inside of the plastic holder*.

   *It should be moving with the shaker and not staying still. Add more buffer or use a different holder if it’s not.

30) Call it a day there and go hunt down a coffee. Fortunately for you, as you decided not to block in 1 hour, you make it to the cafe just before they close.  Go find a friend to vent about your western blot horrors in an attempt to find sympathy.