Anti-C3 antibody (Biotin) (ab48342)

Thank you very much for your reply and information. I just placed an order. I am planning to use the antibody as a detection antibody for a sandwich-type time-resolved fluorescence elisa; if you have any experience on dilutions to start with, I would appreciate an advise.

ANSWER:

I have attached the general Abcam protocol for Sandwich ELISAs, I know you are doing a slightly modified version but I thought it would help. As ab48342 is a purified polyclonal at a concentration at approximately 1mg/ml, I would recommend using 1-2ug of antibody to coat the wells. You may find that you will need to alter it for future experiments, but that would be my recommended starting point.

I am interested in the complement C3 antibody ab48342.

I would appreciate if you can let me know if this antibody can detect

the C3b or C3a part of the C3 molecule.

Thank you in advance and I am looking forward to your respons

ANSWER:

The antibody recognizes both C3a and C3b of C3.

If there is anything else I can help you with, please let me know.

Dear Tech support,

Is there any new data regarding antibodies ab48342, ab48611?

One of my customers wishes to perform "IP" of C3 in Human cells and insists on receiving experimental data, figures or specific protocol for one of these antibodies in order for him to place an order.

Please advice. Regards,

ANSWER:

Thank you for your enquiry.

I have been in touch with the source of these antibodies, and they have kindly provided the following IP protocol. Please note that this may require some further optimization in your own laboratory:

IP protocol is as follows:

1. Have high binding protein A beads (from Peirce) in a column with 3 ml beads bed

2. Wash column with Glycine pH 3.5 (0.1 mM) with 2 column of volume

3. Wash 1x with 1 column volume of PBS 7.4

4. Add 400 ug of antibody to the beads and incubate for 2 hours at room temp with rotating

5. Wash with PBS 2 column volumes

6. Add cell lysates or tissue extract to the column and incubate for 2 hours at room temp.

7. Wash 2 x with PBS

8. Elute the protein and antibody with Glycine pH3.5 (0.1 mM) into the 50 ml conical tube with 250 ul of 1 M tris base as neutralization agent. (collect 25 ml)

9. Concentrate the elution liquid by lyophilization or concentrator.

10. Run SDS to determine the antigen.

This method is for samples with low C3 protein expression. You can also try to coat the antibody to high binding plate and do the IP directly from the plate. The concept is similar but just smaller scale. I am sorry there are no further images to provide on this occasion.

I hope this will be helpful to you. SHould you have any further questions, please do not hesitate to contact us.