SDS-PAGE did not show bands. High ion strength? - (Jul/11/2005 )
Dear all,
I run a SDS-PAGE yestoday. The proteins were extracellular ones that in the supernatant of culture media. The culture media was standard LB media. Before SDS-PAGE, I measured the total proteins concentration is about 3 mg/mL. I load about 20 microgram proteins per well in PAGE gel.
The problem was that there was almost no band show up after staining( I only found one slight band). Since some other samples were run togather with this sample and the bands for those samples showed up, the procedure should be ok. Then, what could be the problem? The LB media was too complex? The ion strength was too high? Or ....
Thenks for your help.
Assuming I am understanding you correctly, you measured protein concentration in you LB media, calculated it be 3mg/ml and ran 20ug of it on a gel.
Since LB consists of casein (a protein) and yeast extract (more proteins), is it possible that those are the proteins you are measuring, and not those secreted by the bacteria?
Assuming I am understanding you correctly, you measured protein concentration in you LB media, calculated it be 3mg/ml and ran 20ug of it on a gel.
Since LB consists of casein (a protein) and yeast extract (more proteins), is it possible that those are the proteins you are measuring, and not those secreted by the bacteria?
It is possible. I run two samples togather. One was induced culture. Another one was non-induced culture. I expected to see a significant bant in the induced sample, which should not show up in the non-induced sample. However, almost no band showed up from both sample! It is strange.
Did you check your bacteria? It is possible the proteins was not secreted into the media.
Was the protein concentration in the media of the induced bacteria higher then uninduced? That would suggest secretion of your protein of interest.
If it was not then.. check your bugs.