Anti-Nanog antibody (ab80892)

Dear Sir/Madam

I do appreciate very much your understanding  and all your efforts to solve the problem. I guess it could be due to a faulty vial.

Therefore, I wish to have a replacement of the same antibody with a different lot.

My best regards    

ANSWER:

Thank you for your understanding in this matter. I have issued a free of charge replacement of your antibody which should be with you tomorrow. The order number is xxxxxxx and the lot: xxxxxxxx.

Having reviewed your protocol there are a few suggestions that I can make that may help to improve the results you have been observing so far:

In order to increase the signal observed for Western blotting I would reduce the level of blocking used. I would suggest blocking for 1 hour at room temperature with 5% milk in TBS-Tween 0.1% but also to try with 1 hour at room temperature with 3% BSA in TBS-Tween 0.1%. I would then try initially incubating the antibody diluted to 1/300 in 1% BSA in TBS-tween 0.1% both for 1 hour at room temperature and overnight at 4°C.

If you are not already doing so I would also strongly suggest using protease inhibitors in your lysis buffer and to make sure the cells are sufficiently lysed to use a buffer such as RIPA. A recipe for which can be found here:

http://www.abcam.com/ps/pdf/protocols/WB-beginner.pdf

This document also contains a more detailed guide of how we would typically recommend preparing the samples.

Hopefully this new batch will prove more successful, however if you do continue to have problems please do let me know. In the meantime I wish you luck with your research.  

Dear xxxx,  

I include images as suggested. Please do observe that in the WB experiment the faint band appearing DOES NOT correspond to the predicted MW.

Best regard

ANSWER:

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. Having reviewed your protocol I can see no reason why the antibody should not be performing. It may be that unfortunately your vial is faulty. I am appreciate the amount of time and effort you have put in to try and get this antibody to produce the results you would like and am very sorry for any inconvenience caused by this. In order to resolve this issue I can offer you a free of charge replacement of this antibody with a different lot, or if you would prefer, another antibody from our catalogue. If this is not satisfactory I can offer a credit note or refund.

Please let me know how you would like to proceed and I will get it arranged as soon as possible. Many thanks for your cooperation in this matter.

Dear Sir/Madam,

I recently bought a rabbit polyclonal antibody to Nanog (ab80892) lot GR40243-2. I have tested several times the same antibody at different dilutions and using both p-phormaldehyde and methanol  fixatives in ICC and IHC experiments using normal and tumoral embryonic tissue (neural and tyroid). Unfortunately we did not get any signal. Moreover we tried also WB experiments but the results were the same with not even a single faint band! We are wondering whether this lot could be not working at all. Could you be so kind to check about that? Hoping to hearing from you soon My best regards   

ANSWER:

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

I have checked back on complaints made against this antibody but there does not seem to be a lot specific problem. It may however be that the vial you have received is faulty. This does not happen frequently but it does happen. I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. If you wouldn't mind completing these I will look at the protocol and see if there is any reason why the antibody may not be performing as expected.

This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.

I am sorry for any inconvenience this problem has caused and hopefully we can resolve the issue as soon as possible. I look forward to receiving your reply. 

Thank you for your reply. J The problem raised from ab19857(Anti-Oct4 antibody) has been resolved by this customer. She tries to decrease the dilution factor of primary antibody from original1/500 to 1/1000 in human HCC sample (1/500 and 1/200 in mouse xenograft sample still works well), and the background is indeed reduced. But now she encounters the similar problem with ab80892 (Anti-Nanog antibody): high background in human HCC sample. She also tries to decrease the dilution factor of primary antibody. The results are : 1/50 works well in mouse xenograft model, but high background in HCC sample; then she tried 1/100 but found no signal in human HCC sample. Would you please help this cutomer provide the suggestion or the solution? Best regards,   regarding the questions: 1) Sample: I assume that your customer has tried this product on xenograft as well as on human tissues. - Could you please specify the human tissue types used for immunostaining?  The sample is from the clinical hepatocarcinoma cancer tissue of patient. - It would also be appreciated if you could confirm the status of the xenograft?  The sample is taken out in 20th day after injection. 2) Blocking: - Please specify what blocking agent was applied?  commercial blocking agent : Cell Marque Background Block Cat NO: 927B-05 Here is the detailed protocol: 1) Sample: I assume that your customer has tried this product on xenograft as well as on human tissues. - Could you please specify the human tissue types used for immunostaining? - It would also be appreciated if you could confirm the status of the xenograft? 2) Blocking: - Please specify what blocking agent was applied? 1) Abcam product code : ab80892 Anti-Nanog antibody (ab80892) lot number : gr40243-2 Purchase order number or preferably Abcam order number : 945992 2) Description of the problem High background in human HCC tissue sample, but in xenograft model it detects well. This customer injects the liver cancer cell lines (such as HuH7, Malhavu and PLC) to the mice subcutaneous tissue. After 20 days, taking the tumor out to conduct IHC-P and the results look good without background. In contrast, in the clinical human HCC tissue sample, the same condition of protocol preformed but high background presented. 4) Sample preparation: Species    Human liver cancer tissue / Xenograft model (human HCC cell line inject to mouse subcutaneous tissue) Type of sample: Fresh frozen sections, perfusion fixed frozen sections, PFA/formalin fixed paraffin embedded sections, cells in culture, other: PFA/formalin fixed paraffin embedded sections Sample preparation Positive control Negative control 5) Fixation step Yes. However, The sample were obtained from the clinical patient sample of the hospital, so the accurate fixation procedure was unable to provide from her. 6) Antigen retrieval method : citric acid pH-6.0 heat a. 121℃ 30mins (for mouse and human sample) b.121℃ 40mins (for human sample) 7) Permeabilization method: Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution buffers? yes Permeabilizing agent and concentration: 0.1% tween20 in wash buffer (wash buffer we using TBS) 8) Blocking agent (eg BSA, serum…): commercial blocking agent : Cell Marque Background Block Cat NO: 927B-05 Concentration, Blocking time 1hour     Blocking temperature : room temperature 9) Endogenous peroxidases blocked? YES (30mins in room temperature) 60ul 30% H2O2, 30ul Methanol, 210ul TBS(total volume: 300ul) Endogenous biotins blocked? NO 10) Primary antibody (If more than one was used, describe in “additional notes”) : Concentration or dilution 1:50(work well in mouse xenograft model, but high background in HCC sample), 1:100(no signal in human HCC sample) Diluent buffer : commercial antibody diluent agent : Cell Marque Cat NO: 936B-0B Incubation time 17 hours in 4℃ 11) Secondary antibody: super sensitive polymer-HRP detection system commercial kit(Biogenex)       Species, Reacts against, Concentration or dilution , Diluent buffer : anti-mouse or anti-rabbit IgG with enzyme polymer in phosphate buffered saline with stablizers and proclin 300 Incubation time 30mins Fluorochrome or enzyme conjugate HRP 12) Washing after primary and secondary antibodies: Buffer 0.1% tween 20 in TBS buffer Number of washes 5mins x 3 times     13) Detection method AEC (Biogenex:QD420-YIK) 14) How many times have you run this staining? 4 times Do you obtain the same results every time? YES 15)What steps have you altered to try and optimize the use of this antibody? Antigen retrieval, primary antibody dilution factor    

ANSWER:

Thank you for getting back to me. I am pleased to hear that the problem with ab19857 has been sorted out after optimization.

Looking through the protocol details you kindly provided for the other antibody ab80892, it seems that the antigen retrieval may be too long (121℃ 30mins and 121℃ 40mins) and the samples are over-treated. This could cause high non-specific background staining.

Generally, 3 minutes is only suggested as a starting point antigen retrieval time. Less than 3 minutes may leave the antigens under-retrieved, leading to weak staining. More than 3 minutes may leave them overretrieved, leading to non-specific background staining and also increasing the chances of sections dissociating from the slides. A control experiment is recommended beforehand, where slides of the same tissue section are retrieved for 1, 2, 3, 4 and 5 minutes before being immunohistochemically stained to evaluate optimum antigen retrieval time for the particular antibody being used.  

I hope this will be useful for you. Should you still have any problem with this antibody after following these suggestions, then please do not hesitate to contact our Technical Department again.      

LOT NUMBER GR40243-2

ORDER NUMBER 905510

DESCRIPTION OF THE PROBLEM

Non-specific band

SAMPLE

mouse ESC lysate

PRIMARY ANTIBODY

Concentration or dilution- 1: 1000

Diluent buffer : 5% skim milk TBST

Incubation time : O/N

Incubation temperature: 4 degree

DETECTION METHODECL

POSITIVE AND NEGATIVE CONTROLS USED

Positive control : sample is positive control

Negative control : MEF

ANTIBODY STORAGE CONDITIONS-20

SAMPLE PREPARATION

Lysis buffer : RIPA

Protease inhibitors: add

Phosphatase:inhibitors: add

Reducing agent: reduced

Boiling for 5 min.

AMOUNT OF PROTEIN LOADED25ug

ELECTROPHORESIS/GEL CONDITIONS

Percentage of gel: 10%

Type of membrane : NC

TRANSFER AND BLOCKING CONDITIONS

Blocking agent and concentration: 5% skim milk

Blocking time : 1hr 30min

Blocking temperature: RT

SECONDARY ANTIBODY

Species: rabbit

Isotype: IgG

Reacts against:

Concentration or dilution : 1: 2000

Diluent buffer: 5% skim milk TBST

Incubation time: 2hr

Incubation temperature: RT

Fluorochrome or enzyme conjugate: HRP

washing Buffer : TBST

Number of washes : 3 times

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

WHAT STEPS HAVE YOU ALTERED?

Blocking buffer: BSA -> Skim milk

ADDITIONAL NOTES

Our user could not detect specific result from your produc. He wants to issue a credit note from you. I attached an image file, please check this for our user. I look forward to hearing from you.

ANSWER:

Thank you for your email. I am sorry to hear that you have been experiencing problems using this antibody. Additionally thank you for sending the image as it was helpful in understanding the problem.

I have checked the protocol which looks fine to me. However by looking at the image I would say there might be problem with the buffers or running voltage.

- Image shows very diffuse band which could be due to slow migration of protein. In this case please increase voltage. Check buffer and ensure it is properly prepared.

- The diffuse band could also be due to problems in sample preparation. Please heat the lysates at 95-100C for 5 minutes.

- The marker bands are also not very well resolved so please check the quality of gel ingredients and prepare fresh buffers.

- Try blocking membrane with 5% BSA solution which in some cases works better than milk.

- Could you please check the secondary antibody also? It looks like you have use rabbit secondary. I would suggest using HRP conjugated anti rabbit secondary antibody.

I hope these suggestions will be helpful. Should you have any other questions please do not hesitate to contact us.