I am new at Western Blotting so unsure what could be going wrong.
It looks like my secondary antibody (Cell Signalling Goat Anti-Rabbit-HRP) is binding to the PDVF membrane in the absence of a primary antibody, giving bands around 100-110kD and 75kD.
I am seeing these bands only in the liver lysate and not in my spleen lysate, which is showing my expected Rabbit Anti-mouse Beta-Actin band if it is added to the filter. I never seen the Beta-Actin band in the liver lysate.
I am currently using 0.05% Tween20-TBS for the washes and secondary antibody dilution, 3% BSA TBS for the blocking and 3% BSA 0.05% Tween 20 TBS for the for the primary antibody dilution. The antibodies are diluted to 1:1000.
Can anyone help me figure out what could be going wrong?
Secondary antibodies are very sticky. Also with the amount of antibody you are using you are likely to get a lot of background. For actin do a 1:5000 to 1:10000 dilution. And back off the secondary antibody too; do 1:5000
Back off your primary and secondary antibodies do 1:5000 primary and 1:10000 for secondary. Actin is abundant so you should get the band. You're probably not getting it because it's a weak signal in the liver extract. Alternatively you could try doubling the loading volume
If you're also using CST's biotinylated molecular weight ladder and anti-biotin HRP antibody to see the ladder, you are likely detecting biotinylated liver proteins with the anti-biotin HRP rather than with the secondary antibody.
If it is the problem of antibody stickness as Sathyen well suggested above, you will see that the bands you see with the secondary antibody alone are also observed with ponceau. This is just antibody getting adsorbed in a patch of membrane rich in proteins. Diluting the antibody will surely help.
Hi Emilie,
First, a picture would realy help to judge what is actualy happening with your blot. However, if you are seeing clear bands then it is very unlikely that your secondary ab is "sticking" to your membrane. If you see bands, you see proteins.
So, if that is the case, your primary ab is reacting with some protein. Could possibly be a cross-reaction, but it can also be a dimer of your protein of interest, or a protein complex, or something else.
Diluting your ab helps when your background is high. If you have a clear background and just see bands on a location you didn't expect, diluting won't help.
So, do share a picture of your blot and depict which bands you did not expect. What species are your samples from, by the way?
Hi,
Thanks everyone for the help.
I was definitely adding too much antibody as my latest result shows that I am still getting signal at 1: 5000.
Alexander I have attached an image if this helps.
On here, both antibodies are at a 1:1000 dilution.
I should not be seeing any bands on the filter on the left side as no primary antibody was added, only the secondary antibody and the ECL substrate.
On the one where Beta-Actin was added, I can the same bands in the liver lysate but I cannot see the Beta-Actin band at 42kD.
It's good that you did a secondary only blot. Like I said earlier, secondary antibodies bind any things that is overly abundant. Any way, what you could do is, maybe cut the blot at the 75Kda position and probe the rest with actin. This way you can also try a much longer exposure of about 5 to 10 minutes to see if anything is coming up at all.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies