I am doing western blots on human striatum for ceruloplasmin but cannot get any bands. I know that the concentration of ceruloplasmin in the brain is quite low. So I've tried using a higher concentration of cell lysate and it still isn't working. My antibody works on IHC and is also supposed to work on WB.
Any ideas on dealing with proteins with a molecular weight of about 120 kDa that are normally in low concentration?
Dear Hailee,
I suppose you already verified extract quality satining your gel with Comassie or, better, the membrane with Ponceau.
Due to the protein size, I would increase blotting time to at least 3h, better o/n at 4°C.
Regarding the Ab you are using, it could recognize a tridimensional complex epitope that does not exist on SDS-PAGE.
To test the Ab you should perform 2 different trials.
1) use a highly expressing protein extract on SDS-PAGE as positive control
2) load the same extract as a DOT blot without any denaturation and reduction
If you get a signal from both experiments it's just a matter of antigen amount. You can try to increase sample amount (up to 80-100 micrograms if you can), block membrane with BSA (generally increase signal intensity) and change ECL reagent using a high sensitivity one.
If you get signal only from experiment 2, you have to change Ab, it doesn't work on Western.
I am sorry if this is obvious, but are you using a positive control?
Based on your question it is difficult to tell if there is something going wrong with your WB or if your concentration is simply too low.
Always make sure you add a positive control (recombinant Ceruloplasmin in your case) to your WB experiment. This should ensure that your experimental protocol is sound. If you do not see bands in your positive control, you know that you need to work on your experimental conditions (like antibody concentration, blocking conditions, chemiluminescence substrate, etc).
If you do see bands on your positive control, you know that you should find a way to increase the concentration of Ceruloplasmin in your sample. Using a higher volume of lysate and a speedvac might be helpfull for this.
Good luck!
Control is critical to amke sure no technical issues are being encountered.
Agree with the above answers
I am using beta actin in every WB. And I am using fluorescent antibodies to do a double stain on my membranes. My beta actin always works like normal, and my transferrin and beta actin double WB always works. But my ceruloplasmin never works. I am not using purified ceruloplasmin, which I should obviously do for control.
I have tried increasing lysate concentration but it still does not pick up a singal thing. I've loaded as much as 80 ug/ well so far.
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