|
A couple quick things:
Take a small amount (1 ml) of your chemiluminescent reagent into a darkroom, and add a microliter of your 2nd antibody. You should see a flash of light. If this works, your enzyme label and luminescence reagent is working.
Then, prepare dilutions of your first antibody, and spot 1 ul of them in a row on some of your transfer membrane. Let that dry, wet the membrane, and wash it. Incubate it in secondary, wash, and develop as usual. If it works, you should see spots of decreasing intensity where you put your first antibody dilutions.
If all this works, it means your luminescence solution and 2nd antibody are working. If you have a pure sample (positive control) of the protein you are probing for, then you can prepare dilution of it and treat it like the first antibody above, except you would incubate with first antibody, wash, second antibody, wash, then develop.
If you see spots of decreasing intensity, then the problem is in your transfer, or there simply isn't enough protein in your sample to see. Staining with Ponceau S can show you if you have protein transferred to your membrane. Spike a sample with your positive control before running it on the gel to see if it shows up when you do the Western. If it does, then you don't have enough protein in your experimental sample to see.
PM me if you need more help.
DK
|
Quick Reply
