I followed the company dilution recommendation and it seems too dilutent. I have to normalize using many dilutions of primary antibodies to reach best results. What dilutions do you use for these antibodies?.
Dear Heba, I usually use Cell Signaling Technology's antibodies diluted 1:1000 in 1% BSA (bovine serum albumin) which gives clear bands always. However if you are getting weak bands just increase the incubation period with the primary antibody. In my case I incubate 2 hours at room temperature on an orbital shaker and then overnight at 4 C. Good luck..!!
If you are using protein lysate than make sure that atleast you use 20-50 microgram of protein per lane. Than Use 1:1000 dilution of each antibody. Try this once. This will giove you an idea that what dilution you use. Depending on the intensity of bands you can increase or decrease the concentration of primary antibody (Caspase 3,9, p53, cyclin A, cyclin D, VDR). When you do not get a band than either you can increase the amount of antibody or if problm persist than you can increase the amount of protein loaded. Finally if you still do not get a band, use different cells from your lab in which the protein in question is well reported. If you do not get a band than change the brand of that antibody
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