Hi
I am doing bacterial Protein SDS PAGE (Laemmli protocol) using bigger assembly but bands broaden as they migrate down.
Solutions compositions
Buffer I: 1.5M Tris-HCl pH 8.8
Buffer II: 0.5M Tris-HCl pH 6.8
30% acrylamide
Sample buffer 4X: 0.25M Tris-HCl pH 6.8, 30% Glycerol, 8% SDS, 10% 2-ME, 0.04% BPB
Running buffer: 25mM Tris, 192mM Glycine, 0.1% SDS
Stacking gel 4% , Separating gel 10%
Bacterial Protein Lysis using Acetone & 1% SDS
Hi Sandeep,
I think the band broadens because you are using a lower concentration of separating gel. At that point on the gel proteins with closely related size shall overlap each other. So I recommend the following:
1- Make your separating gel 12.5%
2- 20 mA per gel (i.e constant ampere and variable voltage)
3- Make sure you fix your gel in acetic acid (i.e the staining and destaining should contain 10 % acetic acid) to prevent diffusion long after electrophoresis has be terminated
Best wishes.
Broadening of bands can be caused by the presence in the sample of a high salt concentration relative to adjacent samples.
Thanks for your advise But
I am not using any salt for lysis. I also used 12% gel running at 100v & staining and destaining solutions contained 10% GAA but getting same results means band broadening.
Ok, try running at a constant ampere as I earlier suggested of 20 mA per gel and making the voltage vary.
The reason for the suggestion above is, it could be that the voltage isn't high enough to move the proteins (concentration) along the gels so you get diffusion as against WORK (i.e Potential diff x distance). If you make the current constant the potential difference shall vary according to the distance between the two electrodes and resistance along the gel.
Yes, when you fix the voltage it meant current shall be variable and you don't want that for it is the main factor that leads to heat generation (I^2R) and consequently diffusion. Most vertical electrophoresis chambers are operated at field strength of 10–20 V/cm for 1mm thick polyacrylamide gels. Just give it a try. If it works fine. But I am positive about it
hi Surah Thanks for your advise
But i also tried to run at 150v & getting same results and second thing is that i am not using urea. I am trying to separate bacterial whole cell lysate.
Hi Surah
I am not using any kit. The protocol for protein isolation was as below
pellet of 5 ml activated culture was incubated for 1 hr in 5mg lysozyme + mutanolysin.
then this incubated culture was suspended in acetone for 10 min on ice and centrifuged at 7000g for 10 min, then supernatant was discarded and 1% SDS was used to break the cells. This crude extract was used in SDS PAGE.
Hello Sandeep,
I also saw that problem, I could tell you that would be related to the concentration of the protein that are running. When it happened to me, have diminished the initial concentration of the protein to run and the problem was solved a little,
Maybe you could try,
I hope to help you.
Hi Natalia
I also tried to run with less protein conc but then band are not visualised by CBB dye.
Hi Sandeep,
are you using a purified protein or are you using total protein in a sample?
Although I believe that the problem of extension of the bands is generally if not concentration of protein I think that might be the running time or the time of the revelation of the bands.
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf
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