Western blot workflow - theory & practice

The western blot is an experiment used to answer questions like - How much protein X does a cell make under different conditions? The basic premise is - take some mix of proteins (such as the “lysate “you get when you break open (lyse) a cell) ⇨ send them traveling vertically though a gel mesh to separate them by size ⇨ trap them in place ⇨ send them traveling horizontally out of the gel onto a membrane ⇨ use antibodies (proteins that recognize specific other proteins) to probe the membrane to see if and how much of a specific protein is there. full version: bit.ly/westernb...
Here’s a general layout of the experiment:
SDS-PAGE - separate proteins by size & trap them in a gel
TRANSFER/BLOT - move the trapped proteins to a membrane that likes to bind proteins - kinda like a “protein duct tape.” The membrane doesn’t feel sticky to your fingers but it does to proteins (but not after you’ve touched it with your fingers - so use gloves and tweezers!)
BLOCK - 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)⠀
WASH, WASH, WASH - wash off non-bound protein⠀
BIND PRIMARY ANTIBODY - Antibodies are little proteins that recognize & bind specifically to specific parts of other things, with the “other thing” being called an ANTIGEN and the “specific parts” being EPITOPES. In a western blot, the antigen is a protein you’re looking for and the epitope is a specific site on that protein. The primary antibody recognizes your specific protein and binds it, so you get your specificity for the thing you’re looking for - yay! but it doesn’t have anything “seeable” about it - boo 😢
WASH, WASH, WASH - wash off non-bound primary antibody⠀
BIND SECONDARY ANTIBODY - this recognizes your primary antibody and has some “detectable” quality like a fluorophore so it will emit light or an enzyme like horse radish peroxidase (HRP) that will convert an uncolored compound you add to a colored compound (chromogenic method) or light (chemiluminescence). It works because antibodies have a unique part that recognizes some antigen and a “generic adapter part” - but that generic adapter part’s only generic for the particular animal that made it (i.e. the adapter part’s slightly different in mice & rats). So you can use things like “goat anti-rat” which is a secondary antibody that uses its unique part to recognize the generic adapter part of a primary antibody made by a rat (and if that rat antibody is using its unique part to bind your protein…)
We use this 2-tiered strategy so that we don’t need “fancy” (detectable) antibodies for every single protein we want to look for. Plus, it increases sensitivity because multiple secondary antibodies can bind each bound primary one so it amplifies the signal. More on antibodies here: bit.ly/antibody...
WASH, WASH, WASH - wash off non-bound secondary antibody⠀
VISUALIZE - detect the detectable using the detectable’s detection method
more on other types of blots (e.g. Southern blots look for specific DNA & northern blots look for specific RNA) bit.ly/342Ynat
longer text version here: bit.ly/westernb...

[@kknight39]

You’re killing it with these lab techniques. As always , lifesaving!

[@thebumblingbiochemist]

Thanks. Happy I could help

[@SwagAli593]

Great presentation. Well explained.

[@thebumblingbiochemist]

Thank you!

[@yifeizuo]

Hi, thanks very much for the video, it helps me understand a lot. I'm not very clear what do you mean at 3:05. WB only tells the presence of one type of protein, so do SDS and what at the same time?

[@thebumblingbiochemist]

Happy it helped! You can do coomassie to see purity

[@junaidrehmani9829]

Pls do some biostatistics videos which would help with data analysis.thanks!

[@thebumblingbiochemist]

I'm going to leave that to the biostats experts, sorry!

[@eduardoduarte1968]

Nice content. Congratulations! Let me ask you a question: 1. Did the following already happened to you? I bought an antibody from a company and the datasheet said it binds to a 62 kDa protein. However, after the blotting I only saw 50 and 100 kDa bands. 2. Could I use the same gel percentage to separate all proteins regardless of the size? For instance, what would happen if I use a 12% gel to separate every protein I'm working with (regardless of high or low molecular weight, just adjusting the running time)? Could I trust that result? Why not? Thank you

[@thebumblingbiochemist]

1. you could be dealing with isoforms and/or degradation products and/or the protein could run differently than the ladder 2. many of the bands would overlap, small proteins might run off, and big proteins might get stuck in the well

[@eduardoduarte1968]

@@thebumblingbiochemistoh, I see. Regarding 2: I should really separate each protein considering its molecular weight and the respective gel percentage, then? There is no universal gel percentage to separate every protein, then?

[@thebumblingbiochemist]

You can use a gradient gel