Protein Sample Loading Buffer
Posted 06 October 2009 - 12:09 PM
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?
Posted 06 October 2009 - 12:43 PM
then heaven will be yours, before you meet your end
Posted 07 October 2009 - 07:13 AM
• 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.
Posted 07 October 2009 - 03:33 PM
- 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