Help with high background (have tried most trouble shooting) - (Sep/21/2012 )

Hi, long time browser first time poster.

I have attached an image of my western blot membrane. Top two membranes are phopho-cMet and bottom two are total c-Met. The exposure is 30 seconds.

I have been following the protocol given on the antibody company's webpage (cell signaling) and other papers have used the same antibodies with clear results. I have also been calling/e-mailing tech support from the company trying to figure out why my background is so high.

Below is my protocol (also cell signaling company's) from transfer to exposure:

- Wet Transfer of 10% gel to PVDF (already activated by methanol and washed in dH20 for 5 min) at 78V for 2 hours.
- Place transfered membrane (no air/methanol/heat drying) in 5% non-fat dry MILK (have tried BSA as well and was told by company to do Milk) TBS-T(0.05% Tween-20) for 1 hr at room temp with agitation.
- wash three times 5 min each in TBS-T
-Incubate in primary 1:1000 5%Milk for total and 5%BSA for phospho TBS-T overnight at 4C (for those who have used the same antibodies and may have suggestions, Cat# 8198 and 3077)
- wash three times 5 min each in TBS-T
- incubate in secondary 1:2000 in 5% Milk for both total and phospho TBS-T 1 hr at room temp.
- wash three times 5 min each in TBS-T
- ECL 1 min
- expose to film.

In addition to this I have tried additional and longer washes after initial ECL/exposure and high salt wash with the same TBS-T(0.05% Tween-20)+0.5M NaCl with not much improvement.

I have tried with freshly made transfer and wash buffers in case of contamination issues. Same membrane probing with ubiquitious ERK total and phospho antibodies show up nicely.

Greatly appreciate any suggestions!!!

Maybe try increasing your primary addition to 1:10,000 and/or decreasing your secondary to 1:5000. Maybe check your antibodies? Also, maybe try using nitrocellulose membranes instead of PVDF?

I second further diluting the secondary - 1:10,000 is a common dilution for the secondary. You also don't need to wash between blocking and primary.

You can also do further washes between primary and secondary and after the secondary but before the ecl.

I will try lower conc. of secondary. as of now I am making new buffers and ordering new AB and stimulant.

before taking images, try to wipe out ECL liquid.

Have you tried to take image one by one per membrane? sometimes, some image which was very light signal could increase background of the membrane which very strong signal. For some reson, the camera try to compensate the signal and try to make all the images to be the most clear.

Tai on Mon Oct 1 15:58:25 2012 said:

I have not tried to wipe out the ECL liquid. Before I expose to film I let the extra ECL to drip down on paper towel. Also I have tried a different lab's ECL so I don't think it's the ECL. The image I have uploaded is the exposed film scanned to the computer. I do not use a camera to take the image directly from the membrane with ECL.

try nitrocellulose membrane unless you know for sure this pvdf membrane is good for ecl westerns

Increase the NaCl in your wash buffer to 300mM or higher. I find this helps get rid of background.

problem solved! it was the damn antibodies.
I have sent the same image to the antibody company.
Hopefully they will offer a new vial of antibodies free of charge.
Thank you everybody though for you help.

Below is blot done with new antibodies.