Hi!! Someone know best method to analyze SUMO1 by western blot? I tried different buffers to extract proteins, different loading buffers, different antibodies and doesnt work :S:S I'm getting mad really, it shouldnt be too difficoult
Do you have sure that your antibody is suitable for denaturated proteins?
yess I think it, I'm using the same ab that I saw in different articles using SDS-Page...they have resoults, me no jajaja
Ok, so, I worked with SUMO-1 for immunoprecipitation and works very well using RIPA buffer (w/o SDS). Did you try this buffer?
Let me know if you need the recipe.
Good Luck!
Thanks so much Bruna, yes I used RIPA buffer suplied with NEM (N-Ethylmaleimide), a specific inhibitor of sumo protease, I used RIPA without it, I used mPER, I used a buffer with urea, I used laemli like loading buffer, I used other loading buffer, I did acrilamide gel with SDS, I used the ab from abcam and I used other ab from CST... and really i dont know what more to do, it doesnt work...what is your positive control?? I want detect endogenous sumo, is not an overexpression.
Thanks again Bruna
Maria, lets for some possibilities:
don't you have a plasmid oo overexpress SUMO-1 in your cells to validate your antibody?
which NEM concentration you are using? NEM was diluted in ethanol?
do you have antibody for SUMO 2/3?
My positive control was SUMO-1 purified from a in vitro sumoylation kit.
Hi Bruna sorry to be late, I have not plasmid, I'm just at the begining of this issue, the NEM concentration I used was 50mM like main articles say and yes I diluted it in ethanol. I have no ab SUMO2/3...I just was trying to test a minimal hypothesis to see if is possible to continue in this way or not...so I cant waste a lot of money at first jajaja
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