I overexpress my candidate gene with tag and I can detect high RNA level but failure in protein level. I did partial expression in tobacco leaf, and only N-terminal is expressed. So we think the protein turn over is too fast to detect in WB. Does somebody have the same experience as me, or can you give me some idea on that? It's really important to check protein phosphorylation for this gene.
You may need to increase the gel density and be careful in transfer the protein to membrane by shortening the transfer time.
Hi, Yong, which protein size do you expect? Which polyacrilamide (PAA) concentration have you used? with which object do you work?
If you protein is too small, it is still possible that protein went out from the gel. In that case you should increase PAA concentration, increasing pH of the separation gel to 9.15 and reduce run time significantly.
How specific you antibody?
You also may try immunolocalization, which give you faster results to compare with WB.
Good luck!
Dear Yong,
I think that, if the protein has a very short half-life, you should try to mark new nascent proteins with radiactive methionine/cysteine and then do immunoprecipitation before gel electrophoresis. I don't know if that approach is possible in your system. Look here:
http://webdoc.nyumc.org/nyumc/files/sun-lab/attachments/CPCB.ch07.Protein%20Labeling.pdf
http://www.arabidopsis.org/comguide/chap_1_plants/8_in_vivo_labelling.html
Best wishes
@Jiucheng He@Taras Pasternak
protein is about 64KD. and I tried 10% or 12% SDS-PAGE already. and I use FLAG antibody in my WB.
Hi, Yong, may be you have the problem with protein isolation? 64 kDa should be in gel with 10 % SDS-PAGE. I would suggest you to use first immunolocalization with the FLAG antibody. Do you work with tobacco? It should be easy do make immuno! For protein phosphorylation you can use this kit: http://www.piercenet.com/browse.cfm?fldID=02051029 Good luck!
Try also histological analysis. Flag antibody normaly function well
Dear Yong Xiang,
are you sure, your full length protein gets translated properly? You said, the n-term was expressed only, what about the c-term part? How do you overexpress your gene? High level overexpression should not be degraded that fast.
Since
Dennis
thanks a lot.
1. I overexpressed my gene infusion with FLAG drived by 35S promoter. I can detect RNA, one primer from FLAG, another primer from gene..
2.I do partial expression. only N term can get protein, C term NO protein detect
3. for protein isolation, I try several different protocol. and I get same result .
Hi, Yong, So, just do immuno in the tobacco leaf with FLAG antibody. It will be much faster than WB, and you omit the protein isolation step and separation step. Good luck!
Hi, Gael, I agree with you, low dose of MG132 (3-5 µM) may help to stabilize protein, if the problem is in protein degradation during lysis and loading.
HI, Gael, I agree with you, too. I will try some inhibitors during lysis.
Hi, Yong, try also immunolocalization!
It will take only 5 hours and you do not need any lysis! For the MG132, you need to treated leaf before protein isolation. Good luck!
HI, Gorm, the gene is from Arabidopsis, and the native express level is quite low, and I overexpressed it use 35S promoter. I already tried as you said, boil my sample before centrifuge. it doesn't work.
Hi, Yong, please, also remember that 35S promoter will give you high mRNA level, but protein level may not increase so much. Have you tried immuno?
not yet. If the protein level is very low, do you think Immonolocalization will works?Now I plan to add inhibitor first. I also think about protein enrichment.
Hi, Yong, immunolocalization should work! It is also important how much cell expressed your protein. In some case you may have few cells with high expression, but the other is very very low, that's why total protein level looking like low. Immuno will help a lot. you may need 5 hours to perform whole procedure. Do you have good secondary antibody binding to DyLight or Alexa 488? Good luck!
Hi, Yong, for the inhinitor, you can use 5- maximal 10 µM MG132. This will have a strong effect.
Good luck!
thanks a lot to everybody for discussion. And I will start do some experiment according your advice. Taras, I may need your help if I decide to do immunolocalization.
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