No protein spots in 2D gel - Silver staining! - (Aug/13/2010 )
Hi, I have that paper, did the same things, however, when comparing methods, found that desalting with G-25 sephadex columns yielded the best results...
If I don't know the exact protein concentration, would that be ok, I f I know it's order of magnitude, at least? In LC-MS/MS analysis, do I have to input the protein concentration exactly?
Oh, another small question if I may... I have been trying out coomassie blue staining now, however, as the protein loads are much higher (100-240 ug of protein by GE recommendations), I have been having some problems with my focusing which I did not have when I used silver staining... I have run a 24 hour program, and my sample and dye have not reached the (+) end of my IPG strip... Is it ok to prolong the focusing at very high voltages for lots of hours? The program I use is:
step - 2 hs - 150 v
step - 2 hs - 300 v
step - 2 hs - 500 v
gradient - 2 hs - 1000 v
gradient - 4,5 hs - 10000 v
step - 4,5 hs - 10000 v
step - about 8-10 hours at 500 v, until I get to the lab and rmeove the strip for SDS-Page run.
Thank you so much again for your help.
Hi
Will you process the sample by 1D SDS-PAGE LC-MS/MS? If you want identify different proteins as much as possible, you may load a more sample amount in 1D SDS-PAFGE. If you want identify the protein spots from 2DE, MALDI-TOF MS might be a better choice.
About your program, how much is the electric current at 10000V, when the step finished. And I suggest 8000 V or 6000 V for 13cm strip. dye have not reached the (+) end, it may indicate the sample is impurities (containing fat, polysaccharide et al. ). The Clean-Up Kit (GE) or TCA- Acetone precipitation may be helpful to sample purification.
rachelhauser on Wed Aug 25 11:44:41 2010 said:
Oh, another small question if I may... I have been trying out coomassie blue staining now, however, as the protein loads are much higher (100-240 ug of protein by GE recommendations), I have been having some problems with my focusing which I did not have when I used silver staining... I have run a 24 hour program, and my sample and dye have not reached the (+) end of my IPG strip... Is it ok to prolong the focusing at very high voltages for lots of hours? The program I use is:
step - 2 hs - 150 v
step - 2 hs - 300 v
step - 2 hs - 500 v
gradient - 2 hs - 1000 v
gradient - 4,5 hs - 10000 v
step - 4,5 hs - 10000 v
step - about 8-10 hours at 500 v, until I get to the lab and rmeove the strip for SDS-Page run.
Thank you so much again for your help.
you should not see the sample reach the end of the ipg strip (and why is there dye in the sample). the proteins will only migrate to the pH where their net charge is zero and should migrate no farther. the second dimension (sds-page) is where the dye should reach the end of the gel.
Actually the dye is bromophenol blue, and is included in the rehydration solution in which I rehydrate my IPG strips... The dye should migrate to the + of the strip, even though proteins might reach their pI later... It is proff that a current is passing through the strip...!
YuFang 7, I will process by both 1D and 2D SDS-Page... I have no idea what proteins exist in my samples. So, I'll follow your suggestions and tell you what happened, if anything! =) Thank you so much!
Best of luck with your experiments.
rachelhauser on Thu Aug 26 08:21:54 2010 said:
Actually the dye is bromophenol blue, and is included in the rehydration solution in which I rehydrate my IPG strips... The dye should migrate to the + of the strip, even though proteins might reach their pI later... It is proff that a current is passing through the strip...!
in that case you can run the first dimension longer. since you are using an immobilized pH gradient you won't have to worry about cathode drift.