Silver staining gels - the biochemistry of how it works

Silver staining is kinda like being a talent scout & agent for D-list celebrities. You want to find them & make them shine. With a silver stain we can make tiny amounts of protein shine - literally! All it takes is a coat of silver (on the protein not the box….) SILVER STAINING is a sensitive all-protein stain that lets you find invisible proteins trapped in a gel. It’s not as popular as Coomassie, but when it comes to tiny amounts of protein, silver staining does the job more well (pictures English teachers cringing - sorry!) blog with full text: https://bit.ly/silver_staining

When I say sensitive, I mean it. It’s ~50X as sensitive as coomassie-based stains, able to detect as tiny an amount as 0.25 ng of protein! (that’s 25-quadrillionths of a gram).

Note: silver stain can also be used to stain for nucleic acids & in some histology stuff - I'm focusing on staining protein gels, but the same principles apply for the most part

note 2: in the video and in-depth discussion I discuss Bio-Rad's Silver Stain Plus Kit because that's what I use (not a paid endorsement) but I talk about what's inside them so you can see if the method you use has similar components.

I revamped & pieced together some silver staining posts that I went into more detail starting starting here: https://bit.ly/2szMg4k

So, how do you find a tiny amount of protein? First you need to get it out of the shadow of all the “big stars” - you want to spread out the proteins, which you can do using one of the most common techniques in biochemistry, especially for protein biochemists -the SDS-PAGE. It’s a way to separate proteins by size using electricity to send them swimming through a gel mesh, which slows down bigger ones more so when you turn off the power they’ll not have traveled as far. Lots more on it here http://bit.ly/sdspageruler

This spreads them out, but they’re still invisible (even the A-listers). Enter the talent scouts - use a protein stain to find where all the actors are. Most of the time we use a stain that binds to all proteins & the most common one is Coomassie Brilliant Blue (CBB) staining, which we went over in detail starting here. It binds non-specifically to proteins and makes them look blue. Much more on it here: http://bit.ly/cbbgelstaining

But this talent scout only finds the low-hanging fruit - it isn’t the most sensitive stain so it won’t detect proteins that are present in really small amounts. The silver stain is really sensitive (sometimes too sensitive as we’ll see…) but it’s more complex - pun not originally intended, but I’ll take it!

Most of the time I use CBB and we even have an instant stain version we use - 1 bottle, stick it on & see your proteins - at least the ones there’s a lot of… http://bit.ly/cbbgelstaining

In contrast (another initially unintended pun…) when silver staining, there are lots of ingredients, bottles, & solutions. BUT there are really only 4 “final” solutions that you bathe the gel in (at least in this protocol… there are probably even more variations on silver stain protocols than there are on Coomassie protocols, which is definitely saying something… )

this protocol uses:
1. fixative
2. water
3. stain
4. stop solution

Regardless of protocol, the basic steps are:
1. fixation: lock the proteins in place & remove interfering compounds
2. sensitization: increase sensitivity & contrast
4. silver impregnation: add silver (in soluble, ionic, form)
5. image development: build up solid silver metal image
6. stop: stop development before excessive background forms

I’ll take you through the steps in more detail, but first here’s the basic premise of the silver stain. The stain solution contains SOLUBLE silver ions (charged molecules). You can’t see them, but they’re there, and they bind to protein in the gel → they get converted to solid silver metal (NOT soluble) → initially you still can’t see it (there’s a “latent image”) → over time it builds up to the point where → you see bands showing the location of protein.

And then you stop it before it starts over-staining the background! Or else you’ll stop seeing bands because your whole gel will just be black…

for some more information:

Chevallet, M., Luche, S. & Rabilloud, T. Silver staining of proteins in polyacrylamide gels. Nat Protoc 1, 1852–1858 (2006). https://doi.org/10.1038/nprot.2006.288
https://www.nature.com/articles/nprot...

Slideshow by Elaine Randall on SlidePlayer https://slideplayer.com/slide/4366092/ which was super helpful for figuring out what’s in the stains