What's inside SDS-PAGE sample loading buffer? And why? From SDS to tracking dye!

The recipe for that blue stuff you mix your protein sample with (and then heat ~90°C for a couple min) before loading it onto an SDS-PAGE gel is pretty simple. It contains:
1. SDS (sodium dodecyl sulfate) - detergent (artificial soap) that detergent that unfolds (denatures) proteins, coats them to keep them soluble, & gives them a negative charge proportional to their length
2. usually added a reducing agent (if you want to use reducing conditions) like BME (β-mercaptoethanol) or DTT (dithiothriotol) to break up disulfide crosslinks - add fresh or at least store samples in freezer once you’ve added it - it will degrade over time
3. Tris - a pH buffer to keep pH stable, typically at 6.8
4. glycerol (or other heavy thing) - helps your sample sink & stay sunk in the well while you load your gel
5. tracking dye (usually bromophenol blue) - lets you track the run’s progress & know when to stop it (warning: it doesn’t show you protein!)
blog form: blog: bit.ly/sds_loa...
You can make (or buy) different stock concentrations of it, given in relative concentration terms (something “X”). If you want/need to load more sample, choose a higher concentration stock loading buffer…For example….
note: I like to prepare extra when possible in case there's a problem & I need to reload or rerun - I make enough for 2 loads and a little extra to give wiggle room for volume loss on pipet tips, etc. For example, I often prepare 36μL & load 15 uL
6X: working concentration is 1 uL per 6 uL total (so add 1uL per 5uL sample) (e.g. 9 uL dye + 27 uL sample)
4X: working concentration is 1 uL per 4 uL total (so add 1uL per 3uL sample) (e.g. 9 uL dye + 27 uL sample)
2X: working concentration is 1 uL per 2 uL total (so add 1uL per 1uL sample) (e.g. 18 uL dye + 18 uL sample)
The recipe we use in our lab for 100mL of 6X is:
5.91 g Tris-HCl pH 6.8
6 g SDS
48 mL glycerol
30 mg bromophenol blue
You can store this at room temperature (RT). Then, before using, add 90 μL of β-mercaptoethanol to 910 μL of this mix - you can freeze this aliquot and it will be good for a while. Note: many protocols use higher [BME] than this (5% is common, but our recipe uses 1.5…)
working concentrations:
80 mM Tris-HCl pH 6.8
1% SDS (w/v)
8% glycerol (v/v)
0.0005% bromophenol blue (w/v)
more on denaturing vs. reducing bit.ly/denatur... ; KZbin: • Denaturing vs. reducin...  
more on SDS-PAGE: bit.ly/sdspager... & • SDS-PAGE theory & prac...  
more on native-PAGE: bit.ly/nativep... & • Variability in protein...  
more random practical lab tips: bit.ly/lab_tri...  
    
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbioc...

[@healthbabe2691]

Your daily short video keeps my brain sharp. Thank you. :) Per my understanding, SDS could be used for cell lysis. If so, would you do a short review for it?

[@thebumblingbiochemist]

Thanks! kzbin.info/www/bejne/f4HJg3efp6ajpa8 bit.ly/homogenization that's how we do it in minipreps too bit.ly/minipreps kzbin.info/www/bejne/i6rHdKV4g5WpeKc

[@healthbabe2691]

@@thebumblingbiochemist You are wonderful. Thank you so much.

[@YasminMonteiro-lj4lk]

Could glycerol negatively affect the running if more of it is added to the sample? I'm using glycerol to protect my samples during storage, but I'm getting trouble with the SDS-PAGE. I wonder if this would be the problem

[@thebumblingbiochemist]

I'm not sure - it probably could if really high concentrations but I don't know

[@YasminMonteiro-lj4lk]

I think i'll try to do with the samples without glycerol to see If It gets better. Thanks for the answer anyway. Your videos are aweasome

[@thebumblingbiochemist]

Good luck! And thank you!

[@ancientk100]

Why do proteins stay with the glycerol and not float away from the well?

[@thebumblingbiochemist]

If you wait long enough they will float away, but basically they're dissolved in a solution with the glycerol and the glycerol is making their surroundings viscous and hard to diffuse out of

[@ancientk100]

@@thebumblingbiochemist thank you!

[@ancientk100]

@@thebumblingbiochemist would microproteins float away more easily if that is the case and higher glycerol is needed? thank you again.

[@thebumblingbiochemist]

I think it should still be fine. They're still way bigger than the dye which doesn't