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immunochemistry


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#1 nmyimliuxianzhong

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Posted 01 September 2010 - 09:45 AM

:D Hi

I want to see per1(flag tag) protein localization after co-expressing CSNK1A (Ha tag).
Primary antibody are Anti-HA in rabbit, Monoclonal ANTI-FLAG, secondary antibody are Anti-mouse IgG 488, 2mg/ml, Anti-rabbit IgG 568, 2mg/ml. green channel is working well but red channel is not working(nothing stained, but DAPI shows nuclei is good). Even I last incubation time for two days for primary and secondary antibody, i'm sure red channel didn't work)
Then I change the primary and secondary antibody:
Primary antibody are Anti-HA in mouse, Anti-Flag in rabbit, secondary antibody are Anti-mouse IgG 568, 2mg/ml, Anti-rabbit IgG 488, 2mg/ml. red channel is still not working(nothing stained but DAPI shows nuclei is good).
I have some experience on GFP,RFP but a few on Alex 568 dye, can you give me any suggestion about Alex 568 dye?
Thank you so much for you suggestion!
Best,
Leo

Primary antibody dilution Second antibody dilution
Anti-HA in rabbit From 1:200 Anti-rabbit IgG 568, 2mg/ml From 1:200(second antibody)
Anti-Flag in rabbit From 1:200 Anti-rabbit IgG 488, 2mg/ml From 1:200(second antibody)
Anti-HA in mouse From 1:200 Anti-mouse IgG 488, 2mg/ml From 1:200(second antibody)
Monoclonal ANTI-FLAG From 1:200 Anti-mouse IgG 568, 2mg/ml From 1:200(second antibody)

#2 laurequillo

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Posted 01 September 2010 - 10:57 AM

So, if I understood the problem is with the antibody. Different proteins do not work with that secondary right?

Just try different antibody concentrations. (if you are sure that you protein is well express). So, use from 1:50 to 1:5000 for example, just to be sure. and try to use a new aliquot of the antibody. Normally for the secondary I just do the incubation for 1h, but you can try longer.
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