Dear All,
I try to make 5x Laemmli buffer (10% SDS, 50% glycerol, 25% 2-mercaptoethanol, 0.02% bromphenol blue and 0.3125 M Tris HCl, pH approx. 6.8.) adapting from Sigma's 2X Laemmli buffer, but I find that is really difficult to make for the following reasons:
1. I try to make it without BME (as some protocol mention to add BME fresh), it seems impossible 50% Glycerol make the solution too thick to mix everything.
2. I try to make about 10ml solution. bromophnol blue is too hard to weight (only 0.02%). I try to make 1%bromophnol blue solution first by diluting to DH2O, but Bromophnol blue doesn't dissolve well in DH2O and leave quite undissolved particulates, and when mix everything with loading sample, it become nearly no color (very faint blue).
If I increase %bromphenol blue in 5x Laemmli buffer for example 1% in total, would it be too high.
Could you reommend how to make this or good recipe for 5x Laemmli buffer?
Thank you
Cheerio,
This may be tricky but here is what we use, prepare, and its perfect for everything we do/done/published. Simply add 5% BME when your ready. That usually stays good for a few months or more at RT or -20C. Re-spike with 1% BME to the working solution if you feel that you need to, e.g. every 2 months. Other components are usually solubilized beforehand in aqueous solution such as the SDS, Tris, and bromophenol blue so to avoid your solubility problem. DTT is not very stable but could also work. BME is supposedly much more stable.
· Prepare 5X Dissociation Buffer (DB) or Gel-Loading Buffer (GLB):
· [“1X GLB = ~1-2% SDS, 10% Glycerol, ~0.02% Bromophenol Blue, 50-125 mM Tris-HCl, pH 6.8, 1% Beta-Mercaptoethanol (BME)” per searches.]
· Make 5X GLB: 5% SDS, 50% Glycerol, 0.1% Bromophenol Blue, 250 mM Tris-HCl, pH 6.8, and, add 5% BME before use.
· For 40 mL:
o 5% SDS: 10 mL of 20% SDS
o 50% Glycerol: 20 mL of Glycerol
o 0.1% Bromophenol Blue: 0.04 g Bromophenol Blue from solid or solution.
o 250 mM Tris-HCl, pH 6.8: 10 mL of 1 M Tris-HCl, pH 6.8
This may be tricky but here is what we use, prepare, and its perfect for everything we do/done/published. Simply add 5% BME when your ready. That usually stays good for a few months or more at RT or -20C. Re-spike with 1% BME to the working solution if you feel that you need to, e.g. every 2 months. Other components are usually solubilized beforehand in aqueous solution such as the SDS, Tris, and bromophenol blue so to avoid your solubility problem. DTT is not very stable but could also work. BME is supposedly much more stable.
· Prepare 5X Dissociation Buffer (DB) or Gel-Loading Buffer (GLB):
· [“1X GLB = ~1-2% SDS, 10% Glycerol, ~0.02% Bromophenol Blue, 50-125 mM Tris-HCl, pH 6.8, 1% Beta-Mercaptoethanol (BME)” per searches.]
· Make 5X GLB: 5% SDS, 50% Glycerol, 0.1% Bromophenol Blue, 250 mM Tris-HCl, pH 6.8, and, add 5% BME before use.
· For 40 mL:
o 5% SDS: 10 mL of 20% SDS
o 50% Glycerol: 20 mL of Glycerol
o 0.1% Bromophenol Blue: 0.04 g Bromophenol Blue from solid or solution.
o 250 mM Tris-HCl, pH 6.8: 10 mL of 1 M Tris-HCl, pH 6.8
For what it's worth, I think that adding BME strictly before use is overkill in most cases. At one place where I worked they used to prepare huge batches of sample buffer + BME which was then stored in small aliquots at 4*C until it was used up. I once checked the performance of fresh vs. 2-year old aliquots using BSA as a control (its mobility changes significantly when reduced) and couldn't find a noticeable difference.
You don't need to worry that much about bromophenol blue. If you're having a hard time dissolving it, just use it less concentrated. The only purpose of bromophenol blue here is to help you locate your sample when you load it on gel. You shouldn't have any problem seeing it even at much lower concentrations.
Bromophenol blue can not be very well dissolved in aqueous buffer or water. You can try to dissolve it in methanol in high concentration (above 1%) and then diluted it in working solution without problem.
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