Blocking is the essential third wheel in any antibody/antigen relationship. Correct blocking buffer can perfect your antibody’s ability to bind its antigen, while bad blocking can make specific antibody binding near impossible. Don’t let bad blocking be a stumbling block in your Western blot experiments – read on to find out what blocking achieves and how to choose the correct blocking buffer for your Western blot.
Why You Need to Block
So you’ve run your gel, done your transfer and now you have a protein-laden membrane just waiting to be probed. Those of us that have been around the Western blot block a few times know that what you do next is critical to prevent non-specific antibody binding and nonspecific signal on your final Western blot. Yep, you guessed it…I’m talking about blocking. The membranes used for Western blots have a high affinity for proteins, which is a good thing – you want your transferred proteins to stick. However antibodies are proteins too, and if not properly blocked from the membrane they will stick too. Not because your antibody has detected its specific antigen but simply because it is a protein. Therefore, it is important to perform blocking on your Western blot membrane right after protein transfer and just before incubation with the primary antibody. The goal of blocking is to cover any membrane surface that doesn’t have any protein already stuck to it, i.e. the membrane space between the lanes, and between your separated protein bands. If not properly blocked these spaces will grab onto your antibody and cause “non-specific binding”. Non-specific binding can cause false positive signals all over your blot – also known as “noise” or “background”, essentially ruining your Western blot. Luckily, blocking buffers can reduce this background noise and improve your signal-to-noise ratio. Sounds simple, right? But like the rest of your Western protocol there are lots of ways blocking can go wrong and make you want to knock someone’s block off.
Pros & Cons of 7 Common Blocking Agents
Your choice of a blocking buffer depends on three things: your antibody, your protein of interest, and your detection system. Here are 7 common blocking buffers and some important tips for how to use them:
1) Skim Milk Powder
The cheapest and most readily available blocking option is skim milk powder. Skim milk powder can either be purchased from a scientific company or over-the-counter at a supermarket. Typically milk powder is used in a 2.5-5% solution. But be forewarned, milk contains a mixture of proteins including phosphoproteins, for this reason milk should not be used if you are using phospho-specific antibodies. That is, unless you want extra background noise, which of course you don’t.
2) Bovine Serum Albumin (BSA)
This is a purified albumin-containing fraction from bovine serum and, along with skim milk powder, is the most common block of choice. Generally BSA containing blocking buffers are at a concentrations of 2-5%. However, researchers often vary the BSA concentration throughout their protocol to use as little as possible because it can be expensive. While this BSA is fine to use with phosphoproteins it can contain contaminating IgG or serum proteins, which can give your non-specific background noise.
3) Fish Gelatin
The use of fish geletin evolved from the use of a previous blocking agent, porcine gelatin. Fish gelatin is purified from the skin of cold-water fish and has the practical advantage of remaining liquid even at cold temperatures. It is used at lower concentrations ranging from 0.1-5%. Unlike BSA or skim milk, fish gelatin doesn’t contain any serum proteins that can cross-react with mammalian antibodies, minimizing background noise. But fish gelatin cannot be used to block with biotin detection systems because it contains endogenous biotin.
4) Normal Serum (eg. fetal calf serum, rabbit serum, goat serum, etc.)
Whole serum, which contains a mixture of proteins, can also be used to block your Western blot membrane. Normal serum is less commonly used as a blocking agent because it offers little practical advantage over skim milk or BSA and it is more expensive. Normal serum also needs to be used at higher concentrations (~5-10%). Like BSA, normal serum contains immunoglobulins and serum proteins that can cross-react with mammalian antibodies.
5) Purified Casein
You could say this one is a chip off the old block, as it is just a purified milk protein. You can use casein where you would use skim milk but be aware that casein is the phospoprotein present in whole milk, so this buffer is also unsuitable if you are trying to detect phosphoproteins.
6) Proprietary Commercial Buffers (eg. Thermo Scientific’s SuperBlock)
These usually contain pure fractions of a mystery proprietary protein. The advantage of these buffers is that you know there are no phosphoproteins, immunoglobulins, albumin or biotin (the bad guys in other blocking buffers), but the downside is that commercial buffers are more expensive. Protocols are specific to each commercial buffer, so consult the manufacturer’s recommendations. A big pro to these blocking buffers is that some of them are quite fast, allowing you to be “quick off the blocks” so to speak and move more rapidly to your detection steps.
7) Polyvinylpyrrolidone (PVP)
PVP is a non-protein alternative to conventional blocking buffers. Although first published in 1993, it’s the new kid on the block in terms of blocking technology. PVP is a water-soluble polymer that readily binds to nitrocellulose and PVDF membrane (the most common Western blot membranes). PVP is generally used at concentrations of 0.5-2% and often in combination with other blocking agents such as purified casein. PVP is particular useful for detecting small proteins, which might be masked by larger blocking molecules like BSA.
What Buffer to Use
So now that you have chosen the best blocking agent for your experiment, what buffer should you dilute it in?…PBST or TBST? Honestly, for most applications it doesn’t matter. Historically, TBST (Tris-Buffered Saline solution with Tween-20 detergent) is preferred for detection of phosphoproteins and should always be used if you are using an alkaline phosphatase conjugate detection systems. But TBST is less stable than PBST (Phosphate Buffered Saline solution with Tween-20 detergent), so if you do use TBST make fresh dilutions frequently and remember to check the pH before use. How do you block your Western blots? Let us know in the comments if you do something different!