I need to isolate a particular kDa protein from a crude antigen (whole cyst). I'm not able to locate that particular kDa protein in SDS gel (10% separating gel of 10X10 size mini gel) stained by coomassie stain or silver staining. But if I blot the gel in NCM and perform ELISA using the corresponding patient serum as primary antibody, I am getting a prominent band in positive samples.
I tried increasing the volume of antigen load, since its a crude protein they get clogged. Also, I tried increasing the concentration of separating gel and the length of the gel (mini to maxi gels) yet I m not able to pick the particular band by staining. I get a smeared region. I need to excise the particular band and carry out downstream processing but I face difficulty in viewing the band in gel.
What do you need the band for? It would be usefull to know in order to make good sugestions.
Have you tried SYPRO Ruby (Life Tech)? It's at least as good as, and probably much more sensitive than, silver staining....
I accept vikram's suggestion, have you added any protein inhibitor to your lysate? if not means it also responsible for protein aggregation and degradation.
Hi Mr. Martiniano, I need to proceed with in gel trypsin digestion and MALDI TOF analysis to identify and characterize the protein.
Hi Mr. Vikram, I havent tried SYPRO protein gel stain so far. Will surely try that out soon.
Thank you so much Mr. Arunas for suggesting me the paper. Hope it will be helpful for my work.
Hi Mr. Mani Gandan, I have used protease inhibitor PMSF during crude antigen preparation for washing the cyst material. Will that be causing any hindrance for staining and visualizing???
Try to staining the gel after washing with distilled water( 2-3 times if required). How much SDS your using for gel or buffer?????. Probably SDS or other chemical in gel running buffer is preventing the staining of your protein by dye....As you might we aware for western blotting follows lots of washing steps...this steps might be getting rid of SDS or other chemical contaminants in gel... Thus your are able to get western blotting results of protein not normal staining....Good luck....
Why dont you try to blot them on PVDF, reveal with antibodies and extract the protein from there. Check this other question https://www.researchgate.net/post/How_to_isolate_proteins_from_PVDF_blotting_paper_for_MALDI-TOF_analysis
Hi. Maybe you need to try to isolate the proteins using a reverse staining with Imidazole-Zn. In our experiencie the procedure is so simple that produced factibility for isolating specific bands for immunization of rabbits (Gonzalez-Malerva et al, Cell Biology International. 2004. 28:885. This strategy was published by Fernandez Patron in several papers. Your final SDS-PAGE is processed by the Zinc-Imidazol reagent and the gels are visualized using a light box inside of a dark room for better observation of the non-stained bands (the gel was completely stained with a pale white color). The process is so rapid, sufficient for taking pictures and the recovery of the bands by cutting them with a scalpel. ....
Article Muscular myosin isoforms of Taenia solium (Cestoda)
I given that you can detect it using an antibody based method why don't you try immuno-precipitation to capture it.
Perhaps you are dealing with a glycoprotein. Have you tried the Periodic Acid Schiff (PAS) staining technique?
Certainly, Antibodies are more sensitive than any of the stains u have used. I also had the same problem during my experiment last week. There should be a lower limit of the amount of protein required for a visible stain in such staining techniques. For eg commassie stained bands of protein less than 10 ng might not be visible but the same protein might show a band when detected by ELISA or WB.
Have you tried a negative stain? As Javier mentioned, these stains are easy to use, and are not restricted by the chemistry of your protein of interest. BioRad makes a couple of negative stains that have worked well for me: http://www.bio-rad.com/en-us/product/negative-stains
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