Im working on yeast secretary protein, my protein was his tagged so i use Anti-his monoclonal antibody for WB, i can see the signal but i got two band on each protein (totally i have two proteins in my supernatant, size is 98KDa and 32 Kda). Two proteins have alpha factor for secretion out from the cell its size about 10 kda, i thought the two band may be because of protein with alpha factor and protein without alpha factor, i also suspect it may be Post translational modification. can anyone suggest me the exact reason for two bands?
HI,
Few possible reasons and solutions.
Target protein has been cleaved or digested Splice variants exist Another protein bearing the same/similar epitope has been detected by antibody. Use a fresh sample which has been kept on ice, add fresh protease inhibitors to the lysis buffer. Try alternate antibody.
Protein may be glycosylated or otherwise modified at one or more amino acid residues. Use enzymes to remove suspected modification returning molecular weight closer to expected Check amino acid sequence and literature.
Dimers, multimers, or protein-protein interactions may be occuring because samples have not been fully reduced and denatured. Add fresh DTT or bME to samples and reheat before repeating experiment Prepare new samples with fresh loading buffer.
Primary antibody concentration may be too high, or there is a cross-reactivity with similar epitopes on other proteins. Use an affinity-purified primary antibody, optimize primary antibody concentration. Try another antibody Check antibody specificity with blocking peptide.
Hope this helps little,
HI,
Few possible reasons and solutions.
Target protein has been cleaved or digested Splice variants exist Another protein bearing the same/similar epitope has been detected by antibody. Use a fresh sample which has been kept on ice, add fresh protease inhibitors to the lysis buffer. Try alternate antibody.
Protein may be glycosylated or otherwise modified at one or more amino acid residues. Use enzymes to remove suspected modification returning molecular weight closer to expected Check amino acid sequence and literature.
Dimers, multimers, or protein-protein interactions may be occuring because samples have not been fully reduced and denatured. Add fresh DTT or bME to samples and reheat before repeating experiment Prepare new samples with fresh loading buffer.
Primary antibody concentration may be too high, or there is a cross-reactivity with similar epitopes on other proteins. Use an affinity-purified primary antibody, optimize primary antibody concentration. Try another antibody Check antibody specificity with blocking peptide.
Hope this helps little,
Hello Marimuthu,
Two bands on each protein means what? Two bands very close to each other? It is possible that while assembling the cassette, you took accessive time during placing and aligning the membrane on the gel and partial protein transfer occurred.
Hi there,
I guess there is a cleavage site between alpha factor sequence and your protein. If the shift between the 2 bands is around alpha factor size it tends to show there is an uncomplete digest
Hi Dr.Liger
Thanks for your answer. there is a cleavage site in my protein, but i can see the two bands in both SDS-PAGE and Western blot. the band shift is about 10-15 KDa. i wonder is there any possibility for this phenomenon.
some of the proteins re-anneal after reduction giving the second band. Get a reduction alkylation kit (BioRad Cat#163-2090 ReadyPrep) and alkylate the proteins after reduction and this should clean up you gels.
Do you have a protocol? If memory serves me well I seem to remember there may be other protocols, but I have been using the BioRad so long I can not remember.
- Badges
- Science method