observed two band for a protein

[sahar sedighzadeh]

Hi all

I have a problem in my work, I expressed a spesific protein with approximate weight 70 KDa in Escherichia coli. but protein samples in SDS-PAGE show two bands close together(70 and 75KDa) that both two bands detected in western blot. I can't justify this phenomenon, is this methylation? if yes how do I prove it? please help me

 

Thanks in advance

best regards

[Kabir Hassan Biswas]

Try all types of post-translational modifications.

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Best wishes,

Kabir Hassan Biswas
Junior Research Associate
Laboratory of Prof. Sandhya S. Visweswariah
Dept. of Molecular Reproduction, Development and Genetics
Indian Institute of Science
Bangalore - 560012
India

[Shet]

Hi,
If your antibody for the detection of expressed protein is against the C-terminal of the protein than
it could be a problem due to translation started also from the next AUG following the start AUG.
It happens many times, especially when researchers clone the gene with starting AUG with a N-terminal-Tag
e.g HIS-Tag. If you have a Tag at N-terminal position and using Tag-specific antibody e.g Anti-HIS, then,
the problem could be something else.

Best wishes

shet

Shet Masih, Ph.D
Post Doctoral Researcher
Department of Microbiology Control
Drexel Institute for Biotechnology & Virology Research
Drexel University College of Medicine
PA 18902, USA

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[sahar sedighzadeh]

Dear Dr.Shet

Hi

Thank you very much for reply. yes my protein has His taq in C-terminal and I detect it by anti His antibody and I don't have any taq in N-terminal position (except GST). I used of pET41a that containing GST, another person in our lab that worked with this vector observed two bands for his protein. whether this is related to GST protein?

 

Best regards.

[Chintan Modi]

1.  Are you cleaving N-term GST-tag?  If so, you might have some extra amino acids post cleavage, which might increase the M.W. of your protein. 
2.  Also, are your purifying your proteins using the GST tag or the his-tag?  If you are using His-tag then there is a possibility that you might be getting contamination from E.coli proteins. This kind of contamination usually occurs if you are doing a large scale purification. 
3. Anit-His antibodies are known to bind non-specifically to several E. coli proteins. 
4. Finally, make sure that you don't have two start codons, one for GST and one for your protein, if you do then there is another reason you might see a high M.W. band?

If you His-tag is good enough for your purification process, then eliminate GST-tag.  Also, make sure you have a very good anti-His antibody and don't concentrate your protein prep prior to metal affinity purification.

So, there could be multiple reasons for detecting two bands.  If you are able to eliminate all these possibilities then you should not see a second band. 

I hope this helps.

Chintan

Chintan Modi
The University of Texas at Austin
Institute of Cell & Molecular Biology
1 University Station
434 Patterson Building
Austin, TX 78712
Phone: (512)475-6424

[sahar sedighzadeh]

Dear Chintan Modi

hi

thaks for your helpful points.

 

Best regards