4 replies to this topic

[biotef]

I am trying to prepare 5X and 6X gel loading buffer( we routinely used 2X but sample volume was getting restricted, we want to load more volume of sample). the composition i follow for 5X is 0.375 mM tris pH 6.8, glycerol 50%, sds 10%, BPB for colour. i slightly heat solution while preparing to dissolve sds. however on storage at RT (22-25º C) we find crystals, probably of sds due to which we cannot pipette solution. is there anything wrong in my composition. how do i resolve this. do i need to heat solution everytime i need gel loading buffer. please guide me on this. thanks

[labtastic]

Normal.  Its not unusual for sds to crash out at that concentration.

 

Microwave your 5-6x stock for a few seconds (5-20s) and it'll be good to use.

 

Stable for years.

[mdfenko]

i've never had 10% sds crash out at room temperature.

 

maybe the vessel or one of your stock solutions is contaminated with a cation that forms a precipitate with sds?

[labtastic]

10% SDS in 50% glycerol (i.e. 5x sample buffer) has always crashed out in every batch I've made for the last 15 years. I assume this is why it's hard to buy 5-6x loading buffer with this formulation. Most commercial 5x recipes that I've seen have either less glycerol or less sds.

 

10% sds in water usually shouldn't crash out, but can on occasion...depends on manufacturer and, as you suggested, probably purity.

 

Non-issue after a few seconds of microwaving tho.

Edited by labtastic, 20 September 2016 - 07:09 PM.

[Missle]

I hate making 5x because of this reason (and getting the SDS into solution) and so purchase Pierce's 5x Lane Marker sample buffers (~$60 for 5mL). It's not too expensive given my level of SDS-PAGE usage and saves time....