Goat anti-Mouse IgM mu chain (HRP) secondary antibody (ab5930)

I finished western blotting experiment yesterday. The expression of CYP1A1 and telomerase in lung cancer cell lines were detected.

There is no signal developed on the telomerase membrane. Multi-bands were developed on CYP1A1 memebrane (Please find attached pictures).

The procedures are described as follows:

1) Sample protein concentration was 20 ug. Same samples were loaded indentically onto two gels. Two gels (10% precast gel) were run together.

2) Membranes transfer was successful, visualised using ponceau red. The membranes was destained completely in TBST. They were incubated separately in blocking buffer (5% non-fat milk+TBST (Bio-Rad)) overnight at 4oC with agitation.

3) Then the membranes were treated with their specific antibodies for 2 hours at 4oC with agitation (CYP1A1 1:500 in blocking buffer; Telomerase 1:500 in TBST + 1% BSA (Sigma A4503)).

4) Membranes were rinsed in TBST, repeated 3x5 minutes.

5) CYP1A1 membrane was incubated with goat anti-rabbit IgG HRP-conjugate (Upstate) (1:5000 in blocking buffer) for 80 minutes at room temperature with agitation. Telomerase membrane was incubated with goat polyclonal to mouse IgM mu-F(ab)2 Fragment (HRP) (AB5930) (1:7500 in TBST + 1% BSA) for 80 minutes at room temperature with agitation.

6) Membranes were rinsed in TBST, repeated 3x5 minutes. Bands were developed using DAB/HRP detection.

In the CYP1A1 detection, the multi bands developed because the primary antibody is polyclonal, but in the HL cell line, there are more bands than the other cell lines, are they non-specific banding? Also, it seems that HL and CD cell lines have much longer bands than the others, do you think that is normal and correct?

Regarding the telomerae detection, was the concentration of first and secondary antibody too low? Why the molecular weight marker (Sigma) is not visualised on this membrane but developed well on the other one (CYP1A1)? Does it because of different dilution buffer used in two detections (milk-CYP1A1 + TBST, BSA-Telomerase +TBST)?

I would be very grateful if you could give me any suggestions. I lookforward to hearing from you soon.

Regards,

ANSWER:

Thank you for your enquiry.

Well done on getting your western blot to work.

I have read your comments and have examined your blot images. I would like ot take each of your antibodies one at a time:

ab3568 - Cytochrome P450 1A1 antibody (ab3568). You have obtained good signal using 20ug of lysate. The blot that you have e-mailed me would have fewer bands were the exposure to DAB have been reduced. We generally advise the use of chemiluminescence (ECL+). This is a more sensitive approach with which you can perform many exposures of the film to the blot. A shortened exposure would have generated significantly fewer bands.

Given that you have detected bands on the blot very easily; to improve the specificity of the antibody and decrease the number of non-specific bands may I suggest that you use the antibody diluted to >1:500 e.g. 1:800 or 1:1000. This should serve to improve the recognition of the antibody for its target.

I think that the reason why you are observing differences in the "length" of the bands and the number of cross reacting bands is due to the fact that the mass of the lysates that you are using are subtly different. The "longer" bands are due to diffusion of the lysates into adjacent areas and it is no consequence that there are more cross reacting bands on account of the larger mass.

May I suggest that you try using the antibody diluted >1:500 and use an ECL or ECL+ detection system (provided you have a dark room), perform different exposure lengths and normalise your lysates so that you can be confident that they are all 20ug. To quantitate the mass of protein you will need to perform a Bradford assay or similar protein quant.

With regards the telomerase, please can you tell me which of the antibodies that you were using e.g.abxxxx. I cannot imagine that the reason that you do not have any bands on the second blot is due to the use of BSA. BSA often gives a better, cleaner more specific blot. Can you tell me whether this blot gave a good Ponceau S staining? Were there any differences in the two blots. The transfer is the most likely problem, but if you tell me the antibody that you used I can check the datasheet to determine whether I can offer any more tips specific to the antibody e.g. dilution etc.

I look forward to hearing from you with further details.

I hope this information helps, please do not hesitate to contact me should you require further assistance.