Rhodopsin in Drosophila is 35kDa membrane protein. Do you know some good homogenization buffer?
In the literature I see that most of the people use Laemmli sample buffer and just simply smash the heads, boil for 5 min, spin and load on the gel. This is strange to me because the are no protease inhibitors etc. I was trying this procedure with no results on the final blot.
Do you know the reason why people use Laemmli buffer instead of standard RIPA ?
Laemmli buffer is a gel loading buffer supplemented with B-mercapoethanol used to denature and seperate proteins for SDS PAGE.
You cannot run a denatured gel with just RIPA extraction, you will need to add laemelli buffer supplemented with B-mercapoethanol and heat denature the proteins for the antibodies to bind to the antigen unless you are running native gel electrophoresis. The SDS will make the proteins migrate in the gel.
You do not always need to add protease inhibitors if you keep your samples cold and denature right away which will get rid of the secondary and tertiary structures of proteases thereby inhibiting them.
Of course, when using RIPA I add laemmli buffer and boil the sample before loading.
This is the blot I did today:
My extraction buffer was: 4% SDS, 1mM EDTA, 75mM Tris/HCL. I removed heads by seeving them in liquid nitrogen and smashed them in buffer. I incubated 5min or 1h RT, spun at 12k rpm. I collected supernatant, added Laemmli buffer, boiled for 5 min and loaded on the gel. As you can see I have no problems in detecting tubulin (right), but there is no rhodopsin band on the left side ;
Here's some questions to help you troubleshoot
Are you using the same secondary antibodies to detect for tubulin and rhodopsin (this lets you know your secondary works)
Can/has anybody shown the rhopdopsin antibody to work in any samples (this lets you know your primary antibody works)
1. Yes I'm using the same secondary antibodys. Primary anti-Rhodopsin and anti-Tubulin are both from mouse.
2. I'm using rhodopsin antibody from hybridoma bank (I ordered it once, after I got no results I ordered it again with the same outcome)(http://dshb.biology.uiowa.edu/4C5) that was shown to work many times, for example:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3244490/
http://www.jneurosci.org/content/25/21/5187.full
I block the nitrocellulose membrane in 5% BSA, incubate overnight with primary antibody, secondary 1h RT and I use ECL kit for developing.
What is really strange for me, is that they all say to do homogenization in Laemmli buffer. Non of them is using RIPA buffer, as far as I know RIPA is quite harsh to get membrane proteins out.
I don't think you'll have a problem using RIPA to extract plasma membrane proteins,
I have used RIPA to extract plasma membrane proteins Caveolin-3 and Cavin-1, but it was a total protein assay.
Hello, I am having the exact same problem/question! We are also using the 4C5 antibody to try to detect Rh1 in fly heads. Did you find out a solution for this?
Did anyone ever resolve this issue? I am using the 4C5 antibody and having similar problems (my Rh1 band is not showing up, but my loading control looks fine. I've tried re-ordering a new batch of antibody, with similar result).
I just tried switching to PVDF (Immobilon-FL), and got it to work right away. Previously, I'd been using nitrocellulose with poor results. Best wishes!
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