Protein Sample Loading Buffer - (Oct/06/2009 )
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?
Considering how viscous 4x loading buffers can be, I suspect that a 10x buffer would be very difficult to pipette.
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.
I use this one as a 6x, though it apparently works at 10x as well:
<*>4ml 20% SDS
<*>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