I'm trying to measure DNA damage levels in human PBMCs using the Abcam anti gamma-H2AX phospho-serine 139 antibody (ab11174). All the images I've seen of gamma-H2AX westerns have a single band, but my results have a 15-kD band which varies in intensity between samples (as expected) plus a lower molecular weight band (see image attached).
Does anyone know what the smaller band is? Any suggestions for how to quantify gamma-H2AX (or DNA damage) from these samples?
WB image that you attached above seems to have several nonspecific bands along with the band of your interest. I doubt regarding the lower molecular band that you are actually concern in the upper strip...are you sure that it is not a dye-front staining of nonspecific band.. seems to be..?
Anyway, the unexpected bands on the Western Blot are normally due to several reasons. The WB image that you have attached looks like a replica of protein profile on gel. There might be several possible reasons for getting such unexpected bands on the WB image.
1. Insufficient blocking of non-specific binding.
2. The primary antibody concentration may be too high (also check the source of PrimaryAb, exposure time and washing buffer & procedure) .
3. The secondary antibody may be binding non-specifically (also check the source & concentration of SecAb, exposure time and washing buffer & procedure).
4. Cross-reaction between blocking agent and primary or secondary antibody.
5. Washing of unbound antibodies may be insufficient.
Since you are using phospho-serine 139 antibody in WB, be careful by avoiding the use of non-fat dry milk or milk in blocking buffer. Milk can specifically react with certain antibodies or detection reagents. Milk contains casein, a phosphoprotein that can react with phospho-specific antibodies. Milk also contains variable amounts of avidin that can interfere with avidin-biotin detection systems. Both of these interactions will result in high uniform background as shown in your image.
Apart from above suggestion, try to increase Primary antibody dilution and reduce the exposure time. On the other hand, block the membrane with BSA overnight to reduce NSB and then check.
Since there are several possibilities, you do not have any other options but to carefully check and rectify the issue in order to get specific signal for phospho-serine 139 only on your WB image.
To quantify, you can use densitometry analysis using Image J software.
Best wishes
The protein could be modified, acetylation, methylation, etc that increases the size it runs at. Check other references for the antibody in the same cell type and see if you see the two bands or one.
We also use the same antibody to detect DNA damage. Here are the things that probably might help you in getting a good band.
Use 12% gel. primary antibody dilution can be (1:3000). secondary (1:5000). if you are using pico ECL, dilute that. Exposure time should not exceed more than 1 min. (15sec, 30sec,45 sec and 60 sec). You will get the sharp single band.
As non specific band is observed so secondary antibody dilution as well as time duration for primary and secondary antibody wash is to be increased. To quantify the band imageJ software can be used.
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