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

Описание: 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: https://bit.ly/sds_loading_dyes

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 https://bit.ly/denaturing_vs_reducing ; YouTube:    • Denaturing vs. reducing - especially in th...    
more on SDS-PAGE: http://bit.ly/sdspageruler &    • SDS-PAGE theory & practice: into the buffe...    
more on native-PAGE: https://bit.ly/nativepageoverview &    • Variability in protein isoelectric points ...    
more random practical lab tips: https://bit.ly/lab_tricks_page  
    
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0 or search blog: http://thebumblingbiochemist.com