Dear,
I take a SDS-PAGE electrophoresis gel. It appears some lines which I don't know what they are. The line connect all wells of gel and they are more than two.
What's it and How to fix my trouble?
Please help me.
It may be keratin contamination, which is a common source of background bands in SDS-PAGE. Make sure you wear gloves when preparing your samples and running buffer.
It may be keratin contamination, which is a common source of background bands in SDS-PAGE. Make sure you wear gloves when preparing your samples and running buffer.
The keratin suggestion makes the more sense, these bands are sample concentration-independent, i.e., regardless how much/little sample you load, these bands show the same intensity.
So it might be keratin, or some other contaminant (what purity substrates are you using to prepare the gels?).
hi.whats your staining? your gel has bubble. do you use gradiant? do you use correct concentration for gel?
if you are reusing the running/ tank buffer you used earlier, then those lines are from contaminated running/ tank buffer(either by protein or bacterial/fungal growth) . I had came across such problem earlier.
It might be helpful if you tell us more about your sample and about which band you want to see and which bands are unwanted. I guess the band in the middle is the only one of interest?
If you have a crude lysate you will always expect to have more then one band, even if you overexpressed only one protein. Especially if you have a sensitive staining and/or you loaded pretty much.
Is it silver stained? It seems you have exactly the sample in all lines. The only difference taking into account the number and position of the bands is the concentration. Some suggestions to be considered:
- Clean crystals with ethanol before to prepare the gel.
- Prepare new buffers.
- Use gloves to prepare the gels
If the protein you are running comes from cell culture, then most probably it is the FBS from the culture media. Did you rinse/wash the cells with PBS before lysing them in protein extraction buffer?
I stained silver. I used dH2O for all experiment.
My sample is cell culture. I centrifuged to collect intracellular protein, not wash, so It has high salt concentration.
Did you wash your cells/plate (if the cells are attached) before you lysed the cells to isolate your protein. Since we use pretty high conc. of FBS (10%- which is really BSA) in the media, if you do not wash your plate with PBS, you will always carry over this protein in your cell extract..When you repeat this expt. , wash you cells/plate throughly with PBS and then proceed for protein extraction..You should see this band (almost)gone
What sizes are the unknown/unwanted bands. FBS/BSA is approx 66kD, keratins can vary 44-66kDa.
Hi Ma Cz
Actually, I didn't know exactly what problem is. But I tried with new chemical component with filtrating by 0.45 um filter. I think it'd caused by some impurity
These are impurities. check your protein preparation and include compounds like sodium carbonate which will help you to remove the lose protein contaminants like ribosomes from the membrane and other non membrane impurities. You can also use protease k to remove the proteases present and stop the process with 1mM PMSF. And include a dialysis process with your buffer or Mili Q which will help in purifying other salt contents.
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