Anti-RanBP9 antibody (ab5295)

We have recently tested the RanBP9 goat polyclonal antibody (ab5295). Unfortunately the detection by WB gave signal only at 4ug/ml (50ug of protein loaded). Therefore 1 vial (100 ug) is the necessary amount to make 25ml of antibody in milk/TBST.

We want to carry on several experiments with RanBP9 antibody, including WB, IC and IP, which will be very expensive given its detection limit. As we intend to buy quite a lot of vials of this antibody, we ask for discount in price and/or higher concentration per vial. We will collaborate with you by providing the pictures obtained with this antibody.

ANSWER:

Thank you for your enquiry.

This antibody is a Fast-Track antibody. It has not been fully characterized in Western blot. As such, we cannot guarantee its use in Western blotting; however, you may be able to earn 1000 Abpoints for being the first scientist to give us conclusive information that this antibody works. Please see the datasheet (I have included a link below) for more details (the "Fast-Track antibody terms of use" link). The Abpoints may be redeemed for vouchers that will offset the purchase price of this antibody.

A few recommendations: Did you incubated with this antibody overnight at 4C? If you incubated at room temperature, it is possible to further dilute the antibody and incubate longer with it to conserve antibody.

Also, did you use a blocking agent in the primary solution? It is possible that the membrane is being overblocked, and that by eliminating the blocking agent in the primary, you will be able to detect your protein at a lower antibody concentration.

In addition, in my lab we always trimmed the membrane and used parafilm to make a smaller vessel for incubating with the primary antibody (just slightly larger than the blot), and made up 10ml of solution. I have also heard of labs using "seal a meal" bags to incubate their blots, which allows you to use only a few mls of solution.

I hope this information helps. Please do not hesitate to contact us if you need anything further.

Could you send human RanBP9 antigen to us?

ANSWER:

Thank you for your enquiry.

The synthetic peptide used to raise ab5295, anti-RanBP9 was GSCAFATVEDYLH, corresponding to C terminal amino acids 717-729 of Human RanBP9. Unfortunately the epitope of the antiserum has not been mapped.

I hope this information helps.

When we ued your product RanBP9 (cat# ab5295, order reference No. 97702; custmer No 2187139.), we could not see any bands on SDS-PAGE gel(Western blot and immunoprecipitation)when blotted with RanBP9 Ab, and could see some bands when blotted with other antibodies(it means that the gel is OK). The cell lysates used are from Hep2G(rat hepatocyte cell line), human hepatocyte cell line and human neuroblastoma cell line. It seems that anti human RanBP9 did not work. Could you show us what is positive control for anti RanBP9 blot and some image about positive bands?

Thanks a lot.

Looking forword to reply.

Sincerely,

ANSWER:

Thank you very much for contacting us regarding ab5295. I am very sorry to hear that you are experiencing difficulty with this antibody.

As you may know, ab5295 is one of our Fast-track antibodies and it is not yet fully characterized. We cannot guarantee that the antibody will work in any application other than ELISA against the immunizing peptide and therefore we are unable to offer a refund if the antibody does not work in your application. However, Fast-Track antibodies are offered at a reduced price.

We would be very grateful for you if you could submit a review via the product datasheet. We have a very special Feedback reward system for Fast-track products: we would greatly appreciate your feedback on this antibody, whether positive or negative.

We will award 950 Abpoints to the first researcher who sends us positive feedback (including an image and details of materials & methods). Conclusive negative feedback that leads us to withdraw the antibody will also be rewarded with 950 Abpoints.

Other useful positive and negative feedback will be rewarded with 50 Abpoints. To find out more about our Loyalty Scheme and Abpoints, please visit our website and look at Your Account panel (left bottom section on our home page) or the Earn Abpoints section on the product datasheet.

All awards are subject to approval by Abcam scientists and provision of suitable data. Please submit feedback to fast-track@abcam.com and would be grateful if you could also attach your Western blot image.

We hope to receive your review very shortly. Thank you for your time and your effort.

BATCH NUMBER 105587 ORDER NUMBER 91976 DESCRIPTION OF THE PROBLEM No signal. SAMPLE rat tissues; rat brain primary culture DETECTION METHOD ECL, ECLPlus ANTIBODY STORAGE CONDITIONS -20?C AMOUNT OF PROTEIN LOADED 50 ug ELECTROPHORESIS/GEL CONDITIONS 10% HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

ANSWER:

Thank you for your enquiry.

ab5295 is currently a fast-track antibody and has not been fully characterized. According to the datasheet there is a 76% identity between the immunogen and the corresponding rat homolog. I would therefore consider it highly possible that the result you are obtaining is a result of a failure of the antiserum to cross react against the rat RanBP9.

However, there are several steps and controls that you could include to fully determine whether this is indeed the case.

1. Include a positive human (e.g. HeLa) cell lysate control (~20ug).

2. Try to use PVDF membrane. Generally, we find enhancement of transfer of proteins on PVDF membranes compared to nitroCellulose membranes.

3. Dilute the antibody 1:500 and incubate the filter overnight at 4 oC to see whether you are obtaining any signal whatsoever.

4. If you have enough material, try to load at least 20-30 ug protein/lane.

Please let us know how you got on using this antibody by leaving an Abreview and in return we will award you 50 Abcam Points, which can be redeemed on a number of rewards (a further 100 Abcam Points will be offered for an image). Should you be the 1st researcher to leave positive or negative (conclusive) data in an Abreview for this product we will award you 1,000 Abcam points.

Please send me the powerpoint file that the author of the enquiry sent in regarding this antibody. I would like to know how it performs in w blots prior to purchasing it. Thank you very much.

ANSWER:

Thank you for your enquiry. I'm afraid that I can't send our customer's blot to you, all of our customer information is confidential. As stated on the datasheet, ab5295 is a Fast-Track antibody; Fast-Track antibodies are immunogen affinity purified and are shown by ELISA to have high titre values against their immunogen. We are currently testing them for use in Western blotting. For more details, please see the Fast-Track antibody terms of use.

So for ab5295, preliminary Western blot experiments gave no signal but low background in whole cell HeLa lysates at up to 1µg/ml.

If you have any more questions, please contact us again.