Hi,
I was preparing 6x laemli buffer with mercaptoethanol as giwen below:
Laemmli's Buffer, 6x
- 1.2g SDS (sodium dodecyl sulfate)
- 0.01% bromophenol blue
- 4.7ml glycerol
- 1.2ml Tris 0.5M pH6.8
- 2.1ml ddH2O
Before use add 1/8th volume of β-mercaptoethanol.
However, when I used it with protein sample, its color was so light. It is to hard to see samples during running of sds-page. Are there anyone has different protocol working good?
Dear Sezer,
This is the recipe I use and works really well, I use a higher concentration of Bromophenol blue:
375 mM Tris-HCl pH 6.8
6% SDS
4.8% Glycerol
9% 2-Mercaptoethanol
0.03% Bromophenol blue
For 25ml:
1.47 g Tris-HCl
1.5 g SDS
1.2 ml Glycerol
Mix and warm to 40 ºC to dissolve the components
Then, add:
2.25 ml 2-Mercaptoethanol
7.5 mg Bromophenol blue
Check pH and add ddH2O to make up to 25 ml
Aliquot and store at -20 ºC
Good luck
Hi Sezer,
our recipe for 6x Laemmli is broadly similar to the above without specifying the bromophenol blue component. I prepare the mixture (including beta-mercaptoethanol in our case), then I take a P1000 pipette tip by hand and the jar of bromophenol blue powder. I dip the tip of the pipette into the BB powder so that some of the powder is transferred into the tip (the bottom 2-3mm of the tip). I then take the tube of Laemmli buffer and immerse the tip into the tube and stir for a few seconds. Over time the buffer will acquire the deep sky blue colour in the top 1-2cm of the tube, at which point, remove the tip, replace the lid and invert the tube. This will not require the majority of the powder from the tip. If not dark enough, repeat. It is important to have enough so your buffer is visible, but not so much that you end up with saturation effects.
Good luck, Robin
Dear Sezer,
This is the recipe I use and works really well, I use a higher concentration of Bromophenol blue:
375 mM Tris-HCl pH 6.8
6% SDS
4.8% Glycerol
9% 2-Mercaptoethanol
0.03% Bromophenol blue
For 25ml:
1.47 g Tris-HCl
1.5 g SDS
1.2 ml Glycerol
Mix and warm to 40 ºC to dissolve the components
Then, add:
2.25 ml 2-Mercaptoethanol
7.5 mg Bromophenol blue
Check pH and add ddH2O to make up to 25 ml
Aliquot and store at -20 ºC
Good luck
You might like to use either Dithiothreitol or Dithioerythritol instead of 2-Mercatoethanol. It is a much more powerful reducing agent for disulphide bridges. A final concentration in the sample buffer of 50mM is more than enough. It also is a more stable reagent than 2-mercaptoethanol and has low volatility so it is much less smelly.
Maria Ibars Serra Thank you for the recipe. What do you set the pH of this buffer to ?
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