I am using 12% gel. Tissue was lysated using RIPA buffer and doing semidry transfer.
On Which tissue you are working? first check wether your antibody is good or not, (by taking cell lysate that has your protein of intrest from cellilne) did you ensure transfer of proteins on membrane after blotting (By ponceau staining)
Prepare your tissue lysate by homogenization in RIPA buffer (50 mM Tris-HCl, pH 7.4 + 1% Triton X-100 + 0.2% sodium deoxycholate + 0.2% sodium dodecylsulfate (SDS) + 1 mM sodium ethylenediaminetetraacetate + 1 mM phenylmethylsulfonyl fluoride + 5 µg/ml of aprotinin + 5 µg/ml of leupeptin). The lysate was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl, pH 6.8 + 12.5% glycerol + 1% SDS + 0.01% bromophenol blue) containing 5% β-mercaptoethanol. (Check whether your antibody will work in reduced condition from the antibody instruction manual).
Best Wishes
Hello sir, I am doing from Mice tissue, antibodies are working and I have done ponceau staining and I can see band after staining. But still i am not able to get bands for housekeeping genes.
Thank you very much sir for your reply
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