I have performed SDS-PAGE on a cell lysate (25ug) prepared with 6X LB and fresh BME followed by boiling for 10 min at 95°C, but still I see signal for different epitopes (all should be around 15-20 kDa) with a band at 70KDa . I am afraid something went wrong in the process because it is unlikely all my antibodies are inespecific (some where tested good by confocal microscopy). What could have been wrong in the protocol? any ideas?
Thanks in advance!!
I have seen this problem when there is contamination with albumin (70 kda weight) or when the tissue expresses endogenous peroxidases (I observed this in brain samples). I suggest running two polyacrylamide gels at the same time. Stain one with comasie and with the other one do the transfer, this will show you that the electrophoresis is adequate and that your proteins are not stuck in the gel. After the transfer, stain the membrane with Ponceau red to see if the transfer is adequate. You can also include a negative control and positive controls for your antibodies to evaluate that the binding is specific. You could stain a membrane with only the HRP-coupled secunadry antibody to rule out nonspecific binding. If you are looking to measure ribosomal proteins it would be interesting to review your lysis and isolation protocol.
Hi there,
Do you use conjugated antibodies for direct detection or primary+secondary antibodies? If the latter, do you use only one secondary or several? Do you get a signal at the expected size too?
Hi Dominique,
I use primary + HRP-conjugated secondary antibody. I only see one band at aprox 70KDa for all epitopes. I am afraid it can be that the denaturation of protein was not proper (besides boiling and BME). The proteins of interest are all ribosomal proteins and histones that have MW between 15-20kDa, can this be a hint?
I have seen this problem when there is contamination with albumin (70 kda weight) or when the tissue expresses endogenous peroxidases (I observed this in brain samples). I suggest running two polyacrylamide gels at the same time. Stain one with comasie and with the other one do the transfer, this will show you that the electrophoresis is adequate and that your proteins are not stuck in the gel. After the transfer, stain the membrane with Ponceau red to see if the transfer is adequate. You can also include a negative control and positive controls for your antibodies to evaluate that the binding is specific. You could stain a membrane with only the HRP-coupled secunadry antibody to rule out nonspecific binding. If you are looking to measure ribosomal proteins it would be interesting to review your lysis and isolation protocol.
Hi there,
Did you try secondary antibody alone as a control ? Such uniform response could result from the non specificity of the secondary antibody. If so you should improve blocking treatment and stringency of the probing.
Is it human tissue lysate or cell line lysates? Did you use any Protein-A beads to deplete lysates?
Whats the percentage of resolving gel? For 15 to 20 kDa proteins you can use a 15% or even 20% resolving gel for precise separation of the protein of your interest.
If you feel the denaturing is not sufficient you can go up to 10 minutes at 95 deg C.
Duirng initial experiments positive and negative controls could be included
If it's non specific binding , it can be avoided by varrying the blocking conditions and the concentration of the primary antibody can be reduced. As Dominique Liger suggested use secondary antibody alone as control.
Unspecific bands in Western are quite common, also depending on the purity of the ABs you are using (are they immune serum, protein A/G purified or antigen affinity purified?) The possibility of unspecific bands may also depend on the antigen that has been used for immunization: Whole protein or a (short!) synthetic peptide? Esp with whole protein antigen, monoclonal or polyclonal origin is another factor.
So, depending on the nature of your antibodies, you need to be more or less concerned about extra bands on your blot. As long as you are able to detect what you are looking for, there is not much to worry about, IMHO.
Edit: Some non-related proteins like keratin from shedded skin and hair sometimes make it into the antigen preps used for immunization and also into the samples for Western Blots...
I'd also try tricine PAGE, it gives better resolution for these relatively small protein
Also, please check the dilution range used for your experiment; sometimes using more than required dilution (i.e., concentrated dilution) could result in non-specific bands. Which antibody did you use by the way ?
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