I normally dont care unless it is not detected in your negative control, but in all your test samples.

This might be albumin. Make sure to wash your cells (tissue) well with cold PBS before freezing them. Albumin is the most abundant protein in serum and many antibodies might nonspecifically interact with it.

Thanks Ahmad. I'm not sure how the tissue (brain) was treated as I'm picking up this project from someone else who is no longer here but albumin does seem to match up pretty well with where the band appears.

I no longer have the paper (and a cursory Pubmed/google search turns out nothing), but I once read that old TEMED stocks sometimes produce a fuzzy band around 70 kDa in Western blots, irrespective of primary/secondary Ab or sample. Is that your case? If it were, try centrifuging the TEMED stock before use, or get a fresh batch.

Alejandro, we don't use TEMED because we use precasts.

Another possibility is keratin contamination. Keratins migrate approximately at that position...

Have you tried your secondaries only on the blot? IT can also be that your secondary HRP interacts directly with the band and so you will see it most of the time.

It also happens to us and we are pretty sure this band corresponds to Albumin. If you are working with cell cultures you should perform a minimum of 2 washes with PBS. If you are working with tissue it is a problem if your band of interest is in the same  MW. You could try to perfuse the animal with saline to eliminate blood contamination and minimize albumin appearance, but you wont avoid some ammounts of it. You could try with albumin depletion kits similar to this one http://www.biotechsupportgroup.com/albuvoid-albumin-depletion-kit-serum-or-plasma for your brain homogenates but keep in mind that you could also lose some of your protein of interest.

good luck!

Have you been detecting anything at that range, as a strong unspecific band to any of your targets? Because if you're reprobing the same blots, my experience is when the antibody is very strong, stripping does not work. If I have an antibody like that, I'll probe with it as a last one.

Have you checked your blots after stripping to see how clear they were? if there is any thing left  over from stripping - your targets and 2nd Abs, the reprobing Abs would bind to the left over things to form a new complex and give you some unexpected bands.

It depends on antibody types. I have been characterizing both monoclonal and polyclonal antibodies from human, mouse and hamster. Linear/continuous/sequential and conformational/native/3D epitopes are critical for antibody binding. WB is unable to detect the 3D one, even native gel may fail if the directed epitope has some protein modification and same goes to ELISA, however, it depends on fixation methods for ELISA. To detect 3D binding, best would be IP and needs confirmation with a linear antibody. Unfixed fresh frozen tissue section (5micron) is a good for conformational one. Another choice is MALDI-TOF/MS-MS for the 3D type. For non-specific bands on SDS-PAGE gel with different species, you need to do absorption assays to see the reduced level of binding. For receptor or extracellular ligand molecules, you can run FACS. So one assay cannot give you the final or confirmatory answer for the nonspecific reactivities. Also keep in mind that concentration of both antigen and antibody is an important regulator for proper binding as they may impose prozone phenomenon. Thanks.

In my experience, aspecific binding of that sort is usually either Albumin or IgG. It depends a bit on what sample you are loading, so a bit more information on your sample preparation may be helpful. Also, can you be a bit more specific on which bands you're seeing on which mass ranges (for example add a picture)?

You may want to deplete your sample first using immunoprecipitation or a depletion kit of some kind to get rid of abundant proteins. Diluting your sample a bit more may also do the trick, but that all depends on how strong or faint the bands are.

Also, in my opinion reprobing blots leads to very dubious results as you can never be certain you have stripped your previous antibody completely. So, it may also be possible that you're seeing carryover from one of your first blots. Does the signal keep constant or does it diminish with each probing step?

Our lab is experiencing a similar problem. We are doing western blots using brain lysates. We see nonspecific bands around 65kD and 120kD on all our blots regardless of which primary antibody we use. After incubation in primary antibody, we used a biotinylated secondary followed by an HRP-streptavidin tertiary.

To diagnose the problem, we ran blots with (1) just the secondary and tertiary, and (2) just tertiary. We saw the nonspecific bands on (1) but not (2). We wondered whether the nonspecific binding of the seondary was due to biotin associating with endogenous proteins, and a google scholar search yielded this paper from 1998 that exactly described our problem: 'Detection of Endogenous Biotin-Containing Proteinsin Bone and Cartilage Cells with Streptavidin Systems' (Praul, et al., 1998)

We haven't resolved our problem yet, though we did find an article that discusses a technique for preventing nonspecific binding from biotinylated proteins: 'Pre-hybridisation: An efficient way of suppressing endogenous

biotin-binding activity inherent to biotin–streptavidin detection system' (Ahmed, et al., 2014).

Good luck!

You should confirm if the incubating with only the secondary antibody you get the band. if not, I would recommend conducting a competitive peptide assay by incubating the primary antibody with the antigenic peptide 20 times more concentrated, to determine if there is any nonspecificity.

@Ahmad Al-Moujahed what do you mean with "before freezing them". Do you Think it will help as well if I rinse my wells with PBS before isolating proteins from them?