Anti-Mad antibody (ab9699)

1. The problem that I am having is that I think the band I am seeing is the wrong size (15kd rather than 25kD)

2. I've been test in whole cell lysates from Jurkat, HT29, U937, and nuclear extract from Jurkat

3. I lyse cells directly in sample buffer (about 500ul in 100mm dish) and load 15ul. Samples are boiled before loading

4. Primary antibody was overnight at 4 degree at 1:1000

5. Secondary anti-goat HRP at 1:2000 at RT for 1 hr

6. Detecting by chemifluorescence - lumiglo

7. Background is not due to secondary and I do block with 5% milk but just for 1 hr

8. I have tried this 3 times with different sets of lysates.

9. I was hoping the U937 cells would be the positive control but I still only see the 15 kd band

10. lot number is 35326

ANSWER:

The originator of this antibody has gotten back to me regarding your comments about ab9699. Ab9699 has not been tested before with Jurkat or HT29 lysates, but a 25 kDa band was seen with U937 lysate.

There are a couple of differences between your protocol and the originator's: 1.They used a different lysis protocol (RIPA buffer see protocol below). They estimated that they were getting protein from 1.4E5 cells onto each lane and if you're using much less than that, then this may be a problem. Wouldn't expect that lysing cells directly into SDS would allow much proteolytic degradation but it is also a possibility. 2. Overnight incubation. They didn't need to incubate overnight to get a good signal with this antibody. It may be that by doing so you are increasing background binding. 3. Detection method. They used ECL plus (Amersham) which is claimed to be much more sensitive that Lumiglo. Following on from the last point about overnight incubation it may be that by dropping the incubation time but upping the sensitivity of detection results could be improved.

Lysis Protocol: Cell pellets were washed with ice-cold PBS. 1 ml of RIPA buffer was added per 1E8 cells and incubated on ice for 20 min, vortexing 2-3 times, briefly. The lysate was aliquotted into 1.5 ml microfuge tubes and centrifuged at 13,000 rpm for 5 min in a microfuge. The supernatant was transferred into clean tubes and its protein concentration was measured with BioRad protein assay. The concentration was then adjusted to 5 mg/ml with RIPA lysis buffer. An equal volume of 2 x SDS sample buffer was then added and the cell lysate was boiled for 5 minutes. Lysates were stored at -80C until use.(RIPA buffer = 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 µg/ml Aprotinin, 5 µg/ml Leupeptin, 1% Triton -100, 1% Sodium deoxycholate, 0.1% SDS).

I hope that these suggestions will be helpful for you and please let me know how it works out.