No or Super Strong Signal - (Apr/12/2016 )

I'm getting bad results with one of the strongest antibody possible, Na+K+ATPase. Here are details:

·         Loaded 30ug of cell lysates

·         10% milk block for 1 hour

·         1:10,000 Na+K+ATPase in 10% milk overnight 4oC

·         6 washes for a total of 30 mins

·         1:2000 anti-mouse secondary for 1.5 hours

·         1 hour TBST rinse with 6 washes

·         Develop using high sensitivity ECL on film

I get a faint signal on my positive Liver control sample after 5 minutes, but absolutely no signal on the cells. Ponceau looked fine. Tested ECL on old B-actin membrane, it was fine. Tested secondary antibody, it was fine. I used a primary antibody solution that worked in the past, no signal on my gel again.

My supervisor does the exact same experiment with the exact same samples but loads 60ug of protein and uses a different aliquot. The signal is so strong it bleaches the film in 1 second.

I’m perplexed. Any suggestions or comments?

have you tried diluting the primary less? maybe 1:5000?

have you tried increasing the sample load to duplicate your supervisor's results?

is this the same lot that worked so well at that dilution in the past?

I ran the experiment with 60ug of protein rather than 30ug of protein and 5% milk blocking rather than 10%. The signal was almost too strong with a 5 second exposure time. Could have been a bad aliquot of primary antibody or too much blocking per protein.

too much blocking wouldn't be an issue unless the antibody is binding to the blocking agent (you would also see increased background in instances where you don't deplete the antibody completely).

you can test for this by changing blocking agent or by dot blotting the blocking agent (and blocking the rest of the membrane with a different agent).

How about your positive control this time? I usually use 5% milk, there was no problem there. Maybe you could try 30ug of the protein again, or dilute the primary antibody and second antibody.