Up till now, there are two kinds of 2x Laemmli sample buffers:
Buffer 1) 65.8 mM Tris-HCl, pH 6.8, 2.1% SDS, 26.3% (w/v) glycerol, 0.01% bromophenol blue
Please refer to the original paper: Cleavage of structural proteins during the assembly of the head of bacteriophage T4
http://www.academia.edu/3313242/Cleavage_of_structural_proteins_during_the_assembly_of_the_head_of_bacteriophage_T4
And BioRad, Cat# 161-0737.
Buffer 2) 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromphenol blue and 0.125 M Tris HCl, pH approx. 6.8
Please refer to Sigma, Cat# S3401. And Cold Spring Harbor Protocols.
Buffer 1) is for optimal band resolution based on BioRad manual.
Does anybody know the advantage of buffer 2)?
Thanks.
http://www.bio-rad.com/en-mx/sku/161-0737-2x-laemmli-sample-buffer
http://www.sigmaaldrich.com/catalog/product/sigma/s3401?lang=en®ion=US
Yes, 2% SDS after dilution is better.
Yes, I think higher concentration of Tris-HCl will keep you extract / lysate pH closer to pH 6.8 and it will be better for protein band resolution
Hi Xianfeng, The Laemmli 2x SDS sample buffer contain 2-ME too. You can use any of the two buffers. I prefer make the buffer for my SDS-PAGE analysis. Some times you need a little more SDS or 2-ME for open completely a protein and improve the migration into the gel.
The advantages of buffer 2) are:
It will give you 2% SDS concentration (preferable) after dilution 2-fold.
It will give you 5% 2-mercaptoethanol (preferable for complete reduction of your proteins under denaturing conditions) after after dilution 2-fold
It will give you 62.5 mM Tris-HCl (more buffer capacity supporting necessary pH 6.8) after dilution 2-fold.
@VIKTOR
In buffer 1), 2% SDS is enough to lyse cells even after dilution (1%). Do you mean 2% SDS after dilution is better?
Tris-HCl concentration in buffer 1 after dilution is 32.9, and in buffer 2 is 62.5. Do you think higher conc. is better for protein bands resolution?
I never think about that before, and now hope to know more.
Thank you both.
Yes, 2% SDS after dilution is better.
Yes, I think higher concentration of Tris-HCl will keep you extract / lysate pH closer to pH 6.8 and it will be better for protein band resolution
Hi there,
The best recipe is the one which is working the best for your experiment!
Buffer 1 formulation is from Biorad manual, and low in SDS (only 1% final). It is not the original in Laemmli paper, which comes with 2% SDS final concentration and TrisHCL 63 mM. Sigma, Abcam formulation stick to the original one
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