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Question (5724) | Recombinant human CCL14 protein (ab9846)

I have a problem with detecting your HCC-1 recombinant protein (Cat# ab9846) with your polyclonal Anti-HCC-1 antibody (Cat#ab9845) by western blotting. Could you help me with my problem? Maybe you could forward this mail to somebody who could help me. My protocol was as follows: I have separated 10 ng HCC-1/lane on a SDS-PAGE. Semi-dry blotting to a Hybond-P PVDF membrane, blocked with non-fat milk, primary Ab: 1 ug/ml anti-HCC1 (Cat#ab9845), Secondary Ab: my normal secondary Ab against rabbit, and detected with Supersignal West Dura substare from Pierce. However, I still do not get any band????? Hope you can help me,

I have checked the datasheet for ab9845 -- Rabbit polyclonal to human HCC 1 and note the antibody can be used at a concentration of 0.1 - 0.2 µg/ml and that the detection limit for hHCC-1 is 1.5 - 3.0 ng/lane. You mention that you have used the antibody at a concentration of 1µg/ml (1:500 dilution) using 10 ng of protein. Strictly speaking, you should be able to detect a a band. My advice would be to run another blot loading more protein on your gel - I'd recommend that you try loading 25ng and 50ng of the recombinant protein and if you still can't detect a band that please get back to us. You could also try using the antibody at a higher concentration but 1:500 should be fine.

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