To differentiate 53, 55 and 57kDa in the same lane (PAGE or Western) - (Jul/12/2008 )
Hi~ Do anyone suggest a gel system (glycine SDS-PAGE/ Tricine gel/ urea gel) or gel % (7.5, 10, 12) to separate 53, 55 and 57kDa protein, which I need to see distinct 3 bands in the same lane.
I have tried a 3% stacking and 7.5% separating glycine SDS-PAGE, and transfer to a 0.45um PVDF membrane. Yet the Western blot result was not conclusive enough. And after some calculations, the mentioned condition would give only 0.1cm different of the above protein sizes. it would be ideal if 0.2cm can be obtained.
Thank you very much!
-tamama-
QUOTE (tamama @ Jul 12 2008, 03:57 AM)
Hi~ Do anyone suggest a gel system (glycine SDS-PAGE/ Tricine gel/ urea gel) or gel % (7.5, 10, 12) to separate 53, 55 and 57kDa protein, which I need to see distinct 3 bands in the same lane.
I have tried a 3% stacking and 7.5% separating glycine SDS-PAGE, and transfer to a 0.45um PVDF membrane. Yet the Western blot result was not conclusive enough. And after some calculations, the mentioned condition would give only 0.1cm different of the above protein sizes. it would be ideal if 0.2cm can be obtained.
Thank you very much!
I have tried a 3% stacking and 7.5% separating glycine SDS-PAGE, and transfer to a 0.45um PVDF membrane. Yet the Western blot result was not conclusive enough. And after some calculations, the mentioned condition would give only 0.1cm different of the above protein sizes. it would be ideal if 0.2cm can be obtained.
Thank you very much!
1. Run it out till just before 50kd marker goes out.
2. Gradient gel
3. Buy one of those long bio-rad gel and apparatus (proteomics people use them extensively and run it out till 50Kd.
4. If you are inputting a lot of protein, the signal may overlap. Use less protein in that case.
..
-cellcounter-
HI
Run Urea gel 10-12 % and see the difference.
Njoy
QUOTE (cellcounter @ Jul 12 2008, 10:20 PM)
QUOTE (tamama @ Jul 12 2008, 03:57 AM)
Hi~ Do anyone suggest a gel system (glycine SDS-PAGE/ Tricine gel/ urea gel) or gel % (7.5, 10, 12) to separate 53, 55 and 57kDa protein, which I need to see distinct 3 bands in the same lane.
I have tried a 3% stacking and 7.5% separating glycine SDS-PAGE, and transfer to a 0.45um PVDF membrane. Yet the Western blot result was not conclusive enough. And after some calculations, the mentioned condition would give only 0.1cm different of the above protein sizes. it would be ideal if 0.2cm can be obtained.
Thank you very much!
I have tried a 3% stacking and 7.5% separating glycine SDS-PAGE, and transfer to a 0.45um PVDF membrane. Yet the Western blot result was not conclusive enough. And after some calculations, the mentioned condition would give only 0.1cm different of the above protein sizes. it would be ideal if 0.2cm can be obtained.
Thank you very much!
1. Run it out till just before 50kd marker goes out.
2. Gradient gel
3. Buy one of those long bio-rad gel and apparatus (proteomics people use them extensively and run it out till 50Kd.
4. If you are inputting a lot of protein, the signal may overlap. Use less protein in that case.
..
-shan-