Hello everyone, I need to detect core protein (25kD) for HCV and HBx (17kD) from HBV by western blot. I already detected but the band are still not as clear as I want. I used a 12% SDS gel, incubation of primary antibody overnight and even tried to optimize the time of blocking (1 hour) but still I cannot get perfect results. Can anyone give me efficient and trustful recommendations to get better detection? Thank you in advance.
Use more sample. You can also play with primary antibody dilution. If you are using ECL, use more sensitive grade ECL.
I detected HBX which had a flag tag. I used blocking 1 hour and normal ECL was enough. I used 750 ul for each reagent. And it worked. I usually use used primary antibody (the same primary antibody used for several experiments, i feeze them), it helps in better detection.
Laoding more samples; decrease the lysis buffer to enhance protein concentration; enhancing incubation time and concentration of primary antibody; using enhance buffer (Thermo. #1862322/2862324). It may be helpful if you use 14% gel.
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