Anti-IGFBP3 antibody (ab4248)

 Hello, I would like to obtain a quote for purchasing amount of the anti-IGFBP-3 Ab Cat. # ab4248 that would be adequate to 400 ug of IgG (I am aware that this prouct is in the form of antiserum). If you could please provide the quote ASAP this would be appreciated.  Thank you.  Best regards,        

ANSWER:

Thank you for contacting us.

We are happy to provide quote for 400 micro-litre of this antibody. The concentration of this antibody was not determined so we will be unable to give you quote for 400 µg. We generally do not determine the concentration of IgG in whole antibody serum.

Let me know if you are interested in getting the quote for 400 µl of this product I will then send you the document soon.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Further to our telephone conversation this morning, please find below three scans of western blots which I have produced over the last week or two relating to your IGFBP3 and IGFBP5 antibodies. They were carried out using the ovarian cancer cell line PE04 and were denatured at 60 degrees for 60min prior to running on a 12% SDS-PAGE gel.

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The first blot above shows IGFBP5 (ab4255) and clearly shows a single band of the right mwt (it should be 31kda).

The second blot shows IGFBP3 (definately ab4248 and NOT ab10733 as I thought - I have the spec sheet) with the priamry antibody titrated down at 10microl/15mL, 5microl/15mL and 2.5microL/15mL in Roche BSA-based blocking agent.

The third blot again shows IGFBP3 (again definately ab4248 and NOT ab10733) at 2.5microl/15mL (different secondary antibody to blot 2) in (i) Roche BSA-based blocking agent (speckly one on left) and (ii) in 5% MARVEL in TBS (clear one on right). Despite blot three having a clear, single band when using 5% MARVEL, the band appears to be of approximately 60-65kda rather than the expected 41kda. The markers appear OK as the IGFPB5 in blot 1 is of the correct size.

Any comments?

Many thanks

ANSWER:

Thank you for your patience in awaiting a response. Having discussed this issue with my colleagues and the originator of the product. The following may explain the route cause of the problem

I have been told that in all cases, the IGFBP's are "sticky" and readily aggregate with themselves or other proteins even in SDS. Another somewhat unusual property that using b-mercaptoethanol is much more effective in preventing IGFPB protein interactions than DTT. I would advise the use of 5% b-mercaptoethanol in the loading buffer instead of the DTT that you have been using previously.

Please let me know how you get on with this.