SDS-PAGE theory & practice: into the buffer and behind the scenes!

Today I ran my 739th SDS-PAGE since I started counting, and I videoed & talked it out for you and added it to my more theory-y talk so you get theory + practical for the price of a free video! SDS-PAGE is a technique we use to separate proteins by size (technically length) by unfolding them, coating them in a detergent (artificial soap) called SDS (Sodium DoDecylSulfate), and sending them traveling through a PolyAcrylamide Gel mesh using Electrophoresis). It's super duper useful, so let's talk about it
And if you want a written & graphics version: bit.ly/sdspager...

[@anhmai9859]

Thanks a lot, Brianna. For me as a visual learner, your design is awesome. I really appreciate it!

[@thebumblingbiochemist]

thanks so much - that's really great to hear!

[@student_remo]

bubbles and shrinks dinky gels are so important! thanks for making SDS-PAGE fun for us 🥰

[@thebumblingbiochemist]

You are so welcome!

[@student_remo]

32:55 For anyone else wondering, she's referring to Coomassie blue dyes by Bio-rad.

[@a_m1350]

How can we distinguish the different proteins and their polymorphisms from each other after the separation.
If, say our sample is cattle milk and we are interested in casein and all it's various polymorphism alpha, beta, kappa etc. Are we going to use the same or different markers to identify the required proteins

[@thebumblingbiochemist]

You would need to do a western blot with antibodies specific for each

[@sbnazar]

Great to see u in youtube 😍

[@mauliksadhu8963]

Hey Brianna thank you for this , but I have a question, if we have proteins with similar molecular weight and we want separate it, how can we do this ? Which technique ?
Sorry for my poor English.

[@thebumblingbiochemist]

Just for looking? That's a tricky one - you can try changing the gel percentage to get better resolution but if they're really similar that might not do the trick. If it's for purifying, you can separate based on charge.

[@mauliksadhu8963]

@@thebumblingbiochemist okay, simply we can run native PAGE right ? Or 2DE ?

[@thebumblingbiochemist]

@@mauliksadhu8963 Definitely 2D electrophoresis could help! I'd totally forgotten about that sorry - haven't done that since undergrad! native PAGE might work but might not depending on how different they run. What's your end goal with it?

[@mauliksadhu8963]

@@thebumblingbiochemist
Nothing like an end goal, I'm an undergraduate student, I just asked for the sake of knowledge..

[@thebumblingbiochemist]

Ah - I see. Great ideas!

[@vikrambhaskar7976]

So from what i understood, you go with SDS page for analysing pure individual proteins but doesn't help if your sample is a multi-protein complex right? Then in that case you'd use native page/BN-PAGE

[@thebumblingbiochemist]

If you're trying to see whether they are a complex, you'd use native page. If you don't care if they're a complex or not, you can still use SDS page, and you should see bands for all of them

[@jyotirmayeetalapatra2119]

Very nice video!
If you don't mind, I wanted to know what buffers you use for high mw and low mw weight proteins as mentioned in the video?

[@thebumblingbiochemist]

Thanks! I don't think I have the recipes on hand sorry - it was in my old lab.

[@prakritigoswami4326]

protocol for preparation of running buffer ? I am looking for protocol online, but I am not sure wether it is neccessary to heat to 190 degrees in order to dissolve TRIS and Glycine in distilled water. I would really appreciate your reply; sincerely master's student (who has to do SDS PAGE for her dissertation)

[@thebumblingbiochemist]

I don't have one sorry - the recipe depends on what type of gel you want to run and I don't think you should have to heat it though but haven't made tris-glycine buffer so am not sure. good luck!

[@captainstyler5650]

Once we've collected DNA, do we need to do PCR before running it on gel electrophoresis, or can we directly run the DNA sample on gel electrophoresis?

[@thebumblingbiochemist]

Depends what you're looking for & what you're starting with. You certainly don't always need to run PCR to look at DNA (can use restriction enzymes etc.) but if you start with genomic DNA or something, that's going to be too massive, and if you start with a wide range of sizes you'll get a smear

[@captainstyler5650]

@@thebumblingbiochemist understood, thank you very much

[@keytravelling]

Good video...btw in which lab you are working??

[@thebumblingbiochemist]

Thanks! I'm in the Leemor Joshua-Tor Lab at Cold Spring Harbor Laboratory

[@ancientk100]

what's the difference between low and high molecular weight buffer?

[@thebumblingbiochemist]

I think these are the recipes: openwetware.org/wiki/Sauer:bis-Tris_SDS-PAGE,_the_very_best

[@ryonbang4718]

Thanks for this video!!!!

[@azharulislam4432]

Donno how to thank you enough

[@thebumblingbiochemist]

knowing I'm being helpful is thanks enough!

[@wesuda]

What an excellent video. I would have loved to watch this when I first started in science!
Also, I almost felt down when you mentioned the amount of SDS PAGE gels that you prepared 🫡😱🫡

[@thebumblingbiochemist]

Thank you!