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:

https://www.youtube.com/watch?v=IDENwOYpGRs&ab_channel=thebumblingbiochemist

What's inside SDS-PAGE sample loading buffer? And why? From SDS to tracking dye! (https://www.youtube.com/watch?v=IDENwOYpGRs&ab_channel=thebumblingbiochemist)

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
*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
Tris – a pH buffer to keep pH stable, typically at 6.8
glycerol (or other heavy thing) – helps your sample sink & stay sunk in the well while you load your gel
tracking dye (usually bromophenol blue) – lets you track the run’s progress & know when to stop it (warning: it doesn’t show you protein!)

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: