Anti-INCENP antibody (ab12183)

I would like to see the western blot used to confirm that this antibody recognises INCENP. The data sheet states that the predicted size of INCENP is 106 Kda, but does not say whether the antibody recognises a band of this size on a western blot?? I find the antibody recognises a single band of 80kDa on whole cell HeLa lysate. I see you have no independent reviews of this antibody and the technical inquiries increase my doubt that this antibody recognises INCENP. It seems unwise to continue to market this antibody as "Anti-INCENP" without better (any?) evidence that it recognises INCENP. I would appreciate any data you have on the performance of this antibody in order that I can gain something from the experiments where I have used it. It may be, for some reason, that the 80kDa band I see is INCENP, running at an unusual size. Currently I am unsure what it is! I realise that I am too late for a refund, but that is of little concern compared to the time that I and others may waste from an antibody that recognises the wrong protein!

Thanks for your help,

ANSWER:

I have received confirmation from the source of ab12183 that the antibody will detect a 106 kDa band using whole HeLa extract. They cannot explain why you are detecting a 80kDa band and hypothesize that this could be a splice variant or a lysed form of INCENP. Unfortunately they were unable to provide us with an image of the blot, I apologise for the inconvenience,

BATCH NUMBER 63014 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM I have some doubt about whether this antibody recognises INCENP in a western blot. When used against total HeLa lysate it gives a single band of 80kDa. This band fails to disappear in lysates of siRNAi-treated HeLa, in which INCENP appears to be knocked down by western and IF with another INCENP antibody. I would be interested to hear how it has been confirmed that this antibody recognises INCENP.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? >10 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

ANSWER:

We have just received the following information from the source of ab12183 who tested the antibody in HeLa nuclear extracts:

1. Extract: 10^7 cells/slab of HeLa nuclear. 2.Wet transfer for 90 minutes at 4'C. 3. Blocking solution: 5%-10% non fat dry milk for 2-24 hours at room temperature. 4. Wash and Dilution buffer: 1% BSA in PBS-T. 5. Primary antibody: this product at 1:5,000-1:10,000 dilutions. Incubation for 120 minutes at room temperature. 6. Secondary antibody: HRP anti rabbit IgG at 1:20,000 dilution. Incubation for 60 minutes at room temperature. 7. Substrate: ECL.

I hope this information helps, please do not hesitate to contact us if you need further assistance,

We have tried all of your suggestions and nothing is working. You suggested:

- "First, I suggest running a positive control." our response - IVP is a positive control

- "you did not mention what dilutions you have tried, but you may need to increase the concentration of the primary. Use a minimum dilution of 1/2000 (recommended when using a whole extract of mouse NIH-3T3 and rat PC12 cells and a chemiluminescent detection reagent)- 1/5000 (recommended when using a nuclear extract of human HeLa cells and a chemiluminescent detection reagent)." our response - we used a dilution of 1/1000

- You may also need to increase the concentration of your secondary antibody, and extend the incubation periods for both the primary and secondary. For example, incubate overnight at 4C with ab12183. our response - we icubated at 4C overnight.

We would like to return the antibody and have it replaced with AB2270.

ANSWER:

Thanks for your email. I can send you ab2270 as a replacement. There's no need to return ab12183. Can you please confirm your shipping address, including phone number?

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER Credit C

DESCRIPTION OF THE PROBLEM No signal or weak signal, no band (not recognizing protein)

SAMPLE IVT and IP

PRIMARY ANTIBODY ab12183 (INCENP antibody)

SECONDARY ANTIBODY Donkey anti-rabbit

DETECTION METHOD ECL+

POSITIVE AND NEGATIVE CONTROLS USED IVT=positive control

ANTIBODY STORAGE CONDITIONS 4?

SAMPLE PREPARATION (MP40) / cocktail / 95? C for 10 minutes

AMOUNT OF PROTEIN LOADED 1microlitre of IVT product

ELECTROPHORESIS/GEL CONDITIONS SDS-PAGE, 15%

TRANSFER AND BLOCKING CONDITIONS Tris + SDS + METH / 200 MA for 1 hour and 15 minutes, 4% milk in TBST

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No

ADDITIONAL NOTES verified the presence of IVT product by phosphoImager using S35

ANSWER:

Thank you for your enquiry, and I'm sorry to hear that you are experiencing difficulty with ab12183. I would like to make some suggestions in order to assist you. First, I suggest running a positive control. For this antibody nuclear/whole extract of human HeLa cells is recommended. Also, you did not mention what dilutions you have tried, but you may need to increase the concentration of the primary. Use a minimum dilution of 1/2000 (recommended when using a whole extract of mouse NIH-3T3 and rat PC12 cells and a chemiluminescent detection reagent)- 1/5000 (recommended when using a nuclear extract of human HeLa cells and a chemiluminescent detection reagent). You may also need to increase the concentration of your secondary antibody, and extend the incubation periods for both the primary and secondary. For example, incubate overnight at 4C with ab12183. If you have any additional questions, please contact us again.

I have tried to use this antibody to stain Hela cells(methanol fixation). It stained the centrosomes clearly at all stages of cell cycle. So far, INCENP is recognized as chromosomal passenger protein. It should localize to the centromere around metaphse, spindle midzone during anaphase. So I don't think this antibody could be used for immunostaining. If you company cann't repeat the supplier's claim, we will have to return the reagent to your company.

ANSWER:

Thank you for getting back to me with an explanation of the staining you would expect with this antibody. I will remove the recommendation that it is suitable for use in IF and refund you for the cost of the vial you purchased from us. (Order number 49851 shipped on 16 August 2004). Thank you again for your feedback.