Western blot blocking solutions

After you blot, you block! And only then can you safely probe your western blot membrane with antibodies!  Western blots use antibodies to detect specific proteins or protein modifications on a membrane (after you’ve transferred the proteins out of a gel onto the membrane). It relies on antibodies specifically binding the thing you’re looking for - and only the thing you’re looking for. The purpose of the block step in a western blot is to prevent nonspecific antibody binding by getting “generic” proteins to bind the parts of the membrane where your protein isn’t before the antibody has a chance to. Your protein’s only at small portions of the protein duct tape, so you need to coat the rest of that sticky membrane with something “generic” like bovine serum albumin (BSA) or milk (really! it’s chock full of proteins)

much more on western blots here:  http://bit.ly/westernblotworkflow ; YouTube: Western blots: the what's, why's, and... & more practical lab tips and tricks here: https://bit.ly/lab_tricks_page     

Unlike the antibodies which we choose for their extreme pickiness, when it comes to proteins, the membrane holding the proteins we send the antibodies out probing for is protein-sticky, but it isn’t picky, so you have to take great care to avoid false alarms!

The membrane is kinda like “duct tape” that’s only sticky for proteins - but it’s sticky for ALL proteins. When you transfer the proteins from the gel to the membrane, your protein sticks to the membrane, but only at the spots where it’s present in the gel (the proteins should be moving straight horizontally because the electric field you generate is straight from back to front).

The antibodies you’re going to use are also proteins, so they think the membrane’s sticky too…so if they stuck it’d kinda defeat the whole point of the western - to detect specific proteins. You use a primary antibody that recognizes & binds to your protein, then a secondary antibody that binds to the primary antibody, amplifies the signal, and, because its conjugated (“permanently” attached) to something detectable, allows you to visualize where the primary antibody bound.

So it’s important that the places where the primary antibody is bound are only where your protein is. If you add the primary antibody without blocking, it’ll still bind your protein, but it’ll also bind all the exposed sticky surface. So you need to first coat the sticky surface with some “boring” protein. By “boring” I mean it won’t interact with any of the antibodies and/or interfere with any detection methods, etc. Basically, you want something that’s there just to de-stickify the membrane so that the whole membrane is coated in proteins - but be so unreactive that it’s like nothing’s there. A couple common options are BSA (Bovine Serum Albumin) and nonfat milk (really!) Milk’s cheap but can have clumping and cross-reactivity problems. You don’t want to use milk if you’re going to use a biotin-based detection system (because milk has biotin) and you also don’t want to use milk if you are probing for phosphorylation because milk has proteins like casein that are phosphorylated.

Be it BSA or milk, we typically dissolve it in a solution containing a low concentration of a detergent. Most commonly, we use Tris Buffered Saline (TBS) with Tween 20 detergent (so TBST). My starting point is 1% nonfat milk in TBS w/0.1% Tween 20. Then if I have background or signal problems I can optimize as needed. Some helpful resources:

G-Biosciences: Western Blot Blocking: Tips and Tricks for Blocking Agents, Posted by The Protein Man on May 30, 2017 https://info.gbiosciences.com/blog/we...

Thermo Fisher, Western blot tips, tricks and troubleshooting: https://www.thermofisher.com/ps/en/ho...

more on other types of blots (e.g. Southern blots look for specific DNA & northern blots look for specific RNA) http://bit.ly/blotcompass  &  northern blots, Southern blots, weste...

more about antibodies: full text: http://bit.ly/antibodytypesanduses  ; YouTube: Lab antibody considerations - monoclo...   

more on detergents: http://bit.ly/detergentsandsoaps  

more about all sorts of things:  #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0 or search blog: http://thebumblingbiochemist.com        
 more on pipetting viscous solutions: blog: https://bit.ly/pipetting_problems ; YouTube: Pipetting viscous liquids - reverse p...