Do you know why my membrane looks like this?
I did this western for
Anti-hnRNP D/AUF1 antibody ab50692, as primary antibody in 1:1000 overnight at 4c and Anti-rabbit IgG HRP linked antibody as secondary antiody in 1:5000 for 1hr
- the wash after the primary was 3 times each 5min with PBS-T (0.05%) and last wash for 5min with PBS,
- the wash after the secondary was 3 times each 20min with PBS-T (0.2%) and last wash for 5min with PBS
- for blocking = I used 5% non-fat dry milk
- PVDF membran
Hi Arwa Saleh Bazighifan
In my experience, this sort of problem comes from the blocking buffer. Black dots can be linked to non-specific binding of antibodies to your blocking reagent. Either filter your blocking reagent or try using 1.5-3 % BSA blocking buffer instead.
If there is no change, consider using nitrocellulose membranes as these typically give less background.
It also looks like your antibody is binding to the molecular weight ladder - add a blank lane between the ladder and your samples to get a better image.
Hope this helps!
Hello
As mentioned by Jack you need to try 3% BSA as the blocking buffer. In addition, try increasing the secondary antibody dilution to 1:15000.
What is the duration of exposure of the X-ray film? Try reducing the exposure time. Also handle the film with hand gloves.
Good Luck.
The problem either lies with the blocking buffer or primary antibody.
If it is the primary antibody there is not much you can do about it. However, it looks like you bought the antibody from abcam and they have a money back guarantee if you are not satisfied and go through trouble shooting with them. Contact them and talk to technical support and show them your blot to see if they can help.
In terms of blocking, I actually believe that non-fat milk is the superior blocking reagent. We use chemcruz milk from santa cruz and it works really well. You can add your antibody in BSA if you want, but I only do that for phosphorylated proteins and still block with 5% milk.
In terms of your milk, is it freshly made and reconstituted well? Milk can settle and the particles can cause the black dots that appear on your blot. Also, if there is any bacteria in the milk that will cause those black spots as they produce a product that chemi will pick up.
Lastly, PVDF definitely has more background than nitrocellulose, but that is more of a universal background as opposed to punctated areas.
If you are using ECL, this might cause the problem. Exposure problems
This question was asked before look at researchgate data
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies