3 replies to this topic

[cancergeek]

Hi Folks,

Quick question. Is it possible to create a sample loading buffer (for western blots) greater than 4X? Such as a 10X loading solution? I've tried searching different vendors but can't seem to find anything thats that concentrated? Is there a reason for not using something that high? or does anyone happen to have a 10X loading buffer recipe?

Thanks!

[gfischer]

Considering how viscous 4x loading buffers can be, I suspect that a 10x buffer would be very difficult to pipette.

[braincow]

I have a recipe for 5X buffer, and yes, it is fairly difficult to pipette unless it's been warmed up in a water bath or heating block.

• 3 mL of 1M Tris-HCl, pH 6.8
• 5 mL of Glycerol, ≥ 99.5%
• 1 g of SDS powder
• 0.05 g of Bromophenol blue
• Bring to 10 mL with ddH2O

Final concentrations (in a 1X solution): 60 mM Tris, 10% glycerol, 2% SDS, 0.1% bromophenol blue.

I'm not sure how you would go about making a 10X buffer. I think it would require a lower final glycerol concentration than this recipe I have since most recipes call for a final glycerol concentration of 10%. If you're having concerns about your final protein concentration not being high enough, maybe you could concentrate your protein sample before running a western.

Edited by braincow, 07 October 2009 - 07:15 AM.

[bob1]

I use this one as a 6x, though it apparently works at 10x as well:

DSL-LOADING BUFFER

• 616mg DTT
  
• 4ml 20% SDS [final conc = 4%]
  
• 969μl 1M Tris pH 6.8
  
• 8ml (10.08g) Glycerol
  
• 800μl 2% Bromophenol Blue in Water

Add H2O UP TO 20ml

Aliquot into 1ml amounts and freeze at -20˚C