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

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

Рет қаралды 12,903

Күн бұрын

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...

Пікірлер: 33

@anhmai9859 3 жыл бұрын

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

@thebumblingbiochemist 3 жыл бұрын

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

@student_remo 5 ай бұрын

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

@thebumblingbiochemist 5 ай бұрын

You are so welcome!

@student_remo 5 ай бұрын

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

@sbnazar 2 жыл бұрын

Great to see u in youtube 😍

@azharulislam4432 8 ай бұрын

Donno how to thank you enough

@thebumblingbiochemist 8 ай бұрын

knowing I'm being helpful is thanks enough!

@a_m1350 2 жыл бұрын

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 2 жыл бұрын

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

@ryonbang4718 2 жыл бұрын

Thanks for this video!!!!

@jyotirmayeetalapatra2119 Жыл бұрын

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 Жыл бұрын

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

@vikrambhaskar7976 2 жыл бұрын

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 2 жыл бұрын

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

@prakritigoswami4326 7 ай бұрын

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 6 ай бұрын

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!

@mauliksadhu8963 3 жыл бұрын

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 3 жыл бұрын

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 3 жыл бұрын

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

@thebumblingbiochemist 3 жыл бұрын

@@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 3 жыл бұрын

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

@thebumblingbiochemist 3 жыл бұрын

Ah - I see. Great ideas!

@captainstyler5650 Жыл бұрын

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 Жыл бұрын

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 Жыл бұрын

@@thebumblingbiochemist understood, thank you very much

@ancientk100 Жыл бұрын

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

@thebumblingbiochemist Жыл бұрын

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

@bastiwala0808 2 жыл бұрын

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

@thebumblingbiochemist 2 жыл бұрын

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

@wesuda 2 жыл бұрын

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 2 жыл бұрын

Thank you!