Anti-beta Actin antibody [AC-15] (ab6276)

Using ab6276, you are most likely detecting a dimer. I would suggest trying the antibody with different samples to ensure that the antibody detects the 42 kDa band.

I'm sorry to hear that you are experiencing difficulties with ab6276. We have gotten a lot of very positive feedback regarding this antibody from customers who have found it to work extremely well. I suggest that you refer to the reviews submitted by these customers to read details of their protocols which will help you out. It sounds like you may be having problems with non-specific bands. Decrease the concentration of the primary antibody and also decrease the primary incubation period. In addition, since you are using a primary antibody of the same species as your tissue (mouse), the secondary antibody may be cross-reacting with endogenous tissue immunoglobulins. Run a secondary antibody control, without the primary, to test this. Also, after normal serum blocking, block endogenous immunoglobulins with affinity purified Fab fragments reactive with the species of the tissue. If you have any more questions or concerns, please contact me again.

We are very sorry to hear that you are having difficulty with this antibody. Ab6276 has been tested in Drosophila but we do not have any information about SF9 insect cells. According to the datasheet, cultured human or chicken fibroblasts were used to characterised this antibody. Therefore we would recommend using this as positive control. If you are still having problem with this antibody, then we would like to ask you to send us a detailed protocol. We will try to do our best to solve the problem.

Antibodies against actin would be fine for this purpose, however I would also suggest using antibodies against one of the enzymes involved in the glycolysis (i.e. LDH, Piruvate-kinase, it depends on the cell types you are testing).

I have searched our database and cannot find any other reports of bands being found at 30-32 kDa. It could be that the antibody is cross reacting with another protein or the band could be degraded actin. However, this antibody is know to be very specific so I'm not quite sure how to explain your results. I suggest that you take a look at the WB images and corresponding protocols on the online datasheet and the reviews from many other researchers who have used this antibody.

If your sample does not contain endogenous mouse immunoglobulin, the secondary antibody will only recognise this primary antibody, and no background staining will result (the secondary is specific for mouse immunoglobulin). A better choice may be to use the directly conjugated version of this antibody, Mouse monoclonal (AC-15) to beta actin (FITC), as this will eliminate the need for a secondary antibody.

I would expect the antibody to work against the peptide itself, however it has not been tested explicitly in this way. The antibody is used very commonly in Western blotting and thus should recognise denatured protein. I am afraid that we don't supply the peptide for this antibody. I don't know why the peptide sequence is different. I can only suggest that the original sequence used was from a variant from the usual actin sequences. I would expect the antibody to bind to peptide with the original sequence, at least weakly. However, I don't believe the epitope has been precisely mapped for this antibody and so I can't guarantee it.

This antibody has not been tested in IP to our knowledge. We would suggest as an alternative for your experiment using the mouse monoclonal to PCNA (clone PC10). This works very well in IP and should serve as a good control.

Unfortunately we do not know which form this antibody reacts with, but we would predict it recognises both forms.

This antibody is used after washing the blot and reprobing it with the AC-15 antibody. The protocol is described in ref. Liao, J., et al., Direct reprobing with anti-beta actin antibody as an internal control for western blotting analysis. Biotechniques, 28, 216-218 (2000).