Anti-MCP1 antibody [ECE.2] (ab8101)

Greetings,
I recently ordered and received this antibody from you all. I am writing
to request some technical assistance as I was unable to detect the protein
of interest in cell extracts which had been shown by ELISA to secrete
large amounts of MCP-1.
My questions are
1. What is the best protocol for western blotting using this antibody?
2. What is the best positive control for its expression.
In summary, our cell extracts were produced by lysis in RIPA buffer
supplemented with protease inhibitors. After protein quantitation, a large
of amount of protein was loaded onto an 8-15% gradient SDS-PAGE, then
transferred to PVDF membrane. The protein transfer verified by Ponceau S
staining. I blocked in 5% Milk/TBST for 1 h, room temp. incubated the
membrane in ab8108 at 1:50 in 1% Milk/TBST overnight at 4C. Secondary was
anti-Rat-HRP at 1:1000 in 1% Milk/TBST for 1 h. We used regular ECL for
detection.

ANSWER:

Thank you for contacting us. I am sorry to hear that this antibody is not providing satisfactory results. The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

For WB,here's the protocol used:

Samples electrophoresed on 15% SDS-PAGE were blotted on nitrocellulose and blocked with PBS/5% low fat dry milk. The blot was incubated with antibody (0.8 µg/ml) for 1 hr at RT.

Your protocol seems like it should have worked as it was pretty similar.

How much protein was loaded per lane?

What was the species/cell line tested?

This clone was used in WB in this reference: Zoja C et al. Renal expression of monocyte chemoattractant protein-1 in lupus autoimmune mice. J Am Soc Nephrol 8:720-9 (1997). PubMed: 9176841

Probably the best positive control would just be an recombinant mouse MCP1 protein, like ab9901.

http://www.abcam.com/JE-protein-Mouse-ab9901.html

Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

Which mouse tissue is suitable as positive control ? is any pre-treatment (e.g. protease, microwave) necessary?

ANSWER:

Thank you for your enquiry.

For a positive tissue control for ab8101, we recommend any vascular tissue, for instance spleen or lung.

For antigen retrieval, we do not have information on which AR treatment, if any, is optimal for this antibody. We suggest trying a heat-mediated citrate pH6.0 AR, as described in the Protocols section of the datasheet:

http://www.abcam.com/index.html?section=ihc&pageconfig=technical&intAbID=8101&mode=problems

You may also want to try a "+/- AR" test as the epitopes some monoclonal antibodies bind are sensitive to heat or protease treatment and may not require any AR for good staining.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

Are there any published protocols using this ECE.2 antibody?

ANSWER:

Thank you for your enquiry. We're currently aware of one publication which featured the use of this antibody:

Zoja C et al. Renal expression of monocyte chemoattractant protein-1 in lupus autoimmune mice. J Am Soc Nephrol 8:720-9 (1997). PubMed: 9176841

We don't have a specific protocol for this antibody, and would recommend referring to our general protocols which can be found by clicking on the protocols tab on the online product datasheet.

Please contact us again if you have any additional questions.

I would like to know about the molecular weight of MCP-1 using Rat monoclonal to MCP-1(ab8101) in Western blotting.

ANSWER:

Thank you for your question regarding ab8101. The molecular weight of predicted mouse MCP-1 is 12 kDa. The expressed protein has a molecular weight of 12-25 kDa. Variable O-linked glycosylation gives rise to the variation in molecular weight.

Customer's question: I would like to know about the molecular weight of MCP-1 using Rat monoclonal to MCP-1(ab8101) in Western blotting.

ANSWER:

The molecular weight of predicted mouse MCP-1 is 12 kDa. The expressed protein has a molecular weight of 12-25 kDa. Variable O-linked glycosylation gives rise to the variation in molecular weight.