In the last post, we talked about the process of performing a Western blot, and a lot of the steps require optimization. Exactly why is that? How can you optimize these steps?
But why?!
Let's start from the beginning. Why do we need to optimize Western blots at all? Western blots rely on antibodies to detect proteins, and antibodies are created in animals. This process involves purifying a protein (or a chemical), injecting the antigen (protein or chemical) into an animal, collecting that animal's serum, and purifying antibodies. This process produces polyclonal antibodies, and the purification process can affect their specificity. For many scientists, raising one's own antibodies is laborious and unnecessary, as many antibodies are commercially available. If an antibody is not commercially available, one can purify a protein and send it to a company in order to produce an antibody.
The above process results in polyclonal antibodies. Monoclonal antibodies can be derived by isolating B cells from stimulated animals, detecting which B cell population is producing the antibody against your antigen, and growing this B cell in culture. Of course, more steps than this are involved, and the previous sentence is a very rough description. Regardless, using this method, one can make virtually unlimited supplies of antibody.
Importantly, no two animals will react to an antigen in the same way - that's the beauty (and difficulty) of the immune system. Thus, no two antibodies are really the same either - unless you have monoclonal antibodies.
To summarize:
We'll go through these one at a time.
The blocking agent
In Western blotting, two blocking agents are typically used: non-fat milk or bovine serum albumin (BSA). Typically, milk is used because it is cheaper than BSA. Typical concentrations of milk range from 5-10% (weight by volunme). However, for antibodies directed against phosphoproteins, BSA must be used because these antibodies will be neutralized by milk.
In addition to which blocking agent to use, one can also optimize the amount of time for blocking and the temperature.
In my personal experience, 5% milk in 2% TBST (Tris-buffered saline with Tween) for 1 hour at room temperature does the trick. If you're in a rush, 30 minutes at 37 degrees can also work.
Detergent concentration
In Western blotting detergents are used to remove excess antibody and to prevent background noise. Generally, a low concentration of detergent is sufficient to clear antibodies binding non-specifically to a membrane. For me, 2% Tween works well, and it can be diluted in Tris- or phosphate-buffered saline. For antibodies with greater background noise, the concentration of Tween can be increased, or the number / length of washes can be increased.
Antibody concentration
Probably the best place to start with optimization with with how much antibody you're using. Naturally, you want to use less antibody (they're expensive, after all!), but it's good to try a range of concentrations. For me, 1 uL of antibody in 1 mL (1:1000) is a good starting point. Some antibodies work at 1:20,000; others work at 1:100. It entirely depends on the antibody, and the manufacturer should provide guidelines for what concentrations to try.
Also, keep in mind that the concentration of your antibody depends on the application: one concentration for Western blotting may not work for immunofluorescence.
Incubation times and temperatures
Yet another step that requires optimization is how long and how hot to incubate your membranes. Sometimes, antibodies work quickly and you cannot incubate for long periods of time because this will result in higher background. However, other antibodies will require longer incubations, even overnight, in order to see any bands. Generally, longer incubations are done at 4 degrees, while shorter incubations (up to a few hours) can be done at room temperature. You'll never know exactly what incubation time to use until you try.
Secondary antibody concentrations and incubations
As addressed above for primary antibodies, the same should be done for secondary antibodies - sounds like a lot of work, no? The good news it that secondary antibodies are generally "well-behaved." For instance, one particular type of secondary antibody uses a concentration of 1:20,000 in milk or BSA for an hour at room temperature. No optimization required if you know what the conditions are! However, if you're working with a new secondary, especially if it's a new species, optimization is your best bet.
Exposure time
One of the last steps to optimize is how long to expose a blot before developing it. Modern technologies have attempted to supplant this step: new chemiluminescent detectors have reduced our need to measure how long to expose our blots. However, for those of us that can't afford this fancy new equipment, we may rely on the good old film and developer. Using this methodology, the amount of time film is exposed to your completed blot will affect how dark your bands are, as well as how much background you. Of course, you want to optimize this step so that your image is clear and not misleading. Overexposing a blot can lead to bands that all look the same; in reality, they might not be if you were to expose your blot less. Additionally, taking several exposures - at both short and long lengths - will give you a range from which to choose.
Conclusions and ideas
The above gives a general guide for how to optimize a Western blot, but by no means is it exhaustive. You can also optimize your developing reagent, for instance. The best bet is to try something and tweak as needed. Western blots are truly an art, and they can be really, really frustrating. However, a little bit of effort in optimization will save you a lot of headache down the road...
But why?!
Let's start from the beginning. Why do we need to optimize Western blots at all? Western blots rely on antibodies to detect proteins, and antibodies are created in animals. This process involves purifying a protein (or a chemical), injecting the antigen (protein or chemical) into an animal, collecting that animal's serum, and purifying antibodies. This process produces polyclonal antibodies, and the purification process can affect their specificity. For many scientists, raising one's own antibodies is laborious and unnecessary, as many antibodies are commercially available. If an antibody is not commercially available, one can purify a protein and send it to a company in order to produce an antibody.
The above process results in polyclonal antibodies. Monoclonal antibodies can be derived by isolating B cells from stimulated animals, detecting which B cell population is producing the antibody against your antigen, and growing this B cell in culture. Of course, more steps than this are involved, and the previous sentence is a very rough description. Regardless, using this method, one can make virtually unlimited supplies of antibody.
Importantly, no two animals will react to an antigen in the same way - that's the beauty (and difficulty) of the immune system. Thus, no two antibodies are really the same either - unless you have monoclonal antibodies.
To summarize:
- Western blots rely on antibodies for detection
- Creating antibodies is technically difficult and involves injecting an animal with an antigen
- Polyclonal and monoclonal antibodies can be produced and they have different uses
- Different animals produce different antibodies
- Animals within the same species will make slightly different antibodies.
Using antibodies
Antibodies are used for many processes in biomedical science: Western blots, immunofluorescence, ELISA, immunoprecipitation... If an antibody works (or doesn't work) for any of these processes, it may (or may not) work for another. Prior to use, antibodies must be tested and optimized.
The steps that require optimization
Let's review the steps in performing a Western blot that require optimization
- Blocking agent
- Detergent concentration
- Antibody concentration
- Incubation times and temperatures
- Secondary antibody concentrations, incubation times and temperatures
- Exposure time
We'll go through these one at a time.
The blocking agent
In Western blotting, two blocking agents are typically used: non-fat milk or bovine serum albumin (BSA). Typically, milk is used because it is cheaper than BSA. Typical concentrations of milk range from 5-10% (weight by volunme). However, for antibodies directed against phosphoproteins, BSA must be used because these antibodies will be neutralized by milk.
In addition to which blocking agent to use, one can also optimize the amount of time for blocking and the temperature.
In my personal experience, 5% milk in 2% TBST (Tris-buffered saline with Tween) for 1 hour at room temperature does the trick. If you're in a rush, 30 minutes at 37 degrees can also work.
Detergent concentration
In Western blotting detergents are used to remove excess antibody and to prevent background noise. Generally, a low concentration of detergent is sufficient to clear antibodies binding non-specifically to a membrane. For me, 2% Tween works well, and it can be diluted in Tris- or phosphate-buffered saline. For antibodies with greater background noise, the concentration of Tween can be increased, or the number / length of washes can be increased.
Antibody concentration
Probably the best place to start with optimization with with how much antibody you're using. Naturally, you want to use less antibody (they're expensive, after all!), but it's good to try a range of concentrations. For me, 1 uL of antibody in 1 mL (1:1000) is a good starting point. Some antibodies work at 1:20,000; others work at 1:100. It entirely depends on the antibody, and the manufacturer should provide guidelines for what concentrations to try.
Also, keep in mind that the concentration of your antibody depends on the application: one concentration for Western blotting may not work for immunofluorescence.
Incubation times and temperatures
Yet another step that requires optimization is how long and how hot to incubate your membranes. Sometimes, antibodies work quickly and you cannot incubate for long periods of time because this will result in higher background. However, other antibodies will require longer incubations, even overnight, in order to see any bands. Generally, longer incubations are done at 4 degrees, while shorter incubations (up to a few hours) can be done at room temperature. You'll never know exactly what incubation time to use until you try.
Secondary antibody concentrations and incubations
As addressed above for primary antibodies, the same should be done for secondary antibodies - sounds like a lot of work, no? The good news it that secondary antibodies are generally "well-behaved." For instance, one particular type of secondary antibody uses a concentration of 1:20,000 in milk or BSA for an hour at room temperature. No optimization required if you know what the conditions are! However, if you're working with a new secondary, especially if it's a new species, optimization is your best bet.
Exposure time
One of the last steps to optimize is how long to expose a blot before developing it. Modern technologies have attempted to supplant this step: new chemiluminescent detectors have reduced our need to measure how long to expose our blots. However, for those of us that can't afford this fancy new equipment, we may rely on the good old film and developer. Using this methodology, the amount of time film is exposed to your completed blot will affect how dark your bands are, as well as how much background you. Of course, you want to optimize this step so that your image is clear and not misleading. Overexposing a blot can lead to bands that all look the same; in reality, they might not be if you were to expose your blot less. Additionally, taking several exposures - at both short and long lengths - will give you a range from which to choose.
Conclusions and ideas
The above gives a general guide for how to optimize a Western blot, but by no means is it exhaustive. You can also optimize your developing reagent, for instance. The best bet is to try something and tweak as needed. Western blots are truly an art, and they can be really, really frustrating. However, a little bit of effort in optimization will save you a lot of headache down the road...
