Anti-KDM4C / GASC1 / JMJD2C antibody (ab85454)

Here are the picture of my WB and the answers to the survey.


1) Abcam product code ab

ab85454 (GR33581-2) and ab56759 (GR60796-1)



3) Description of the problem



ab56759 works in IF but I am unable to make it works in WB.

Ab85454 works neither in IF, nor in WB.

4) Sample preparation: Cumulus cells and embryos, whole cell lysates

Lysis buffer : SDS-PAGE or Laemmli 2X

Protease inhibitors: No

Phosphatase inhibitors No

Reducing agent

Boiling for ≥5 min? Yes, 9 minutes

Protein loaded ug/lane or cells/lane : 35ug

Positive control : HeLa (ab85454), HepG2 (ab56759)

Negative control : Nothing



5) Percentage of gel : I have tried 10%-8%-6%

Type of membrane Nitrocellulose

Protein transfer verified Yes, with rouge de ponceau

Blocking agent and concentration ECL blocking agent 5% or 5% milk in TBST 0.1%

Blocking time 1h30

Blocking temperature RT



6) Primary antibody (If more than one was used, describe in “additional notes”) :

Concentration or dilution : Both tested 1:500

Diluent buffer TBST 0.1% with ECL blocking agent 5% or milk 5%

Incubation time : O/N

Incubation temperature: 4°C



7) Secondary antibody: HRP goat anti-mouse for ab56759 and HRP goat anti-rabbit for ab85454

Concentration or dilution : Very good secondary antibodies, diluted 1:100 000

Diluent buffer TBST 5% milk

Incubation time 1h

Incubation temperature: RT



8) Washing after primary and secondary antibodies:

Buffer

Number of washes 5 washes 5 minutes



9) Detection method Pellicule



10) How many times have you run this staining? 5 times

Do you obtain the same results every time? Yes

What steps have you altered to try and optimize the use of this antibody? Dilution of the antibody. Blocking agent. % of the gel. Transfer time.
Best regards,

ANSWER:

Thank you for your message and for getting back to me with further details. It is much appreciated.

I am sorry to hear you are disappointed with our products. I appreciate the time you have spent on these experiments in the laboratory and it is disappointing the results have not been successful.

As our Abpromise indicates, in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.

After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions:

1) Tested species:


It would be interesting to know the species of the embryos used in these studies to make sure that the cross-reactivity of the antibody does match with the sample species.

2) Expression levels:

The expression of KDM4C / GASC1 / JMJD2C varies in different section and age of the embryos. You may wish to visit this website for further details:

http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Hs.709425

3) Lysis buffer:

In order to prevent any unwanted degradation of the protein, it is recommended to use a lysis buffer (i.e. RIPA) which contains a mixture of different proteinase inhibitors (i.e.aprotinin, leupeptin, pepstatin A). The recipe for this buffer can be found on our website at:

http://www.abcam.com/index.html?pageconfig=resource&rid=11379#A1

4) Blocking:

Try to reduce the blocking time to 1 hr at RT (or overnight at 4oC) since too stringent/long blocking can reduce/eliminate the signal.

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

As we wrote in a previous email:

we test all the suggestions you gave us, but unfortunately we were unable to get better results in two of the three antibodies we have.

So, for PKN1 (ab47535), we detect specific labelling after the use of your suggestions, although I should remind you that Abcam recommend a dilution of 1/1000 – 1/5000 for IHC-P (see datasheet) and we needed to use 1/500 to get the right signal.

The remaining two antibodies, KDM4C (ab85454)and USP22 (ab71732)still have unspecific labelling and/or no detectable signal even with the suggestions you made.

We don't know if you have any other solution to give us, or if we should apply for the refund.

That is the reason why we only asked alternative products for ab85454 and ab71732.

Please let me know if you need any aditional information.

Thank you for your attention.

Best regards,

ANSWER:

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will provide us with vital information for our monitoring of product quality. I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful fromab85454 and ab71732. Reviewing the details, I am sorry there are no further tips to provide on this occasion to help improve the results. I can suggest you have regrettably received a bad vial.

I apologize for the inconvenience and as requestedam pleased to provide the alternativefree of charge replacements in compensation. I have issued the following free of chargeorder:

Order number:1088302
1 X vial ab123900
1 X vial ab4812

To check the status of the order please contact our Customer Service team and reference this number.

Please note that the free of charge replacement vials are also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

Regarding PKN1 antibody ab47535, I am pleased this is now working for you. I understand your concerns, it is regrettable thatthere were some initial difficulties with this. Please note that any concentrations of dilutions stated on the datasheet are a guideline only, and this will sometimes require further optimization for individual experiments and sample types.

I wish you the best of luck with your research. If you require anything further, please do not hesitate to contact us.

Once we are really interested in those specific proteins, and you didn't suggest any more optimization steps, we would like to replace our products by:


Anti-KDM4C / GASC1 / JMJD2C antibody [5B9] (ab123900)



Anti-USP22 antibody (ab4812)

Do you think that these two will replace the ones we bought with better and faster results? As we already spent a lot of time optimizing the previous antibodies.

Thank you for your attention,

Best regards,

ANSWER:

Thank you for your response.

I have checked our system which stores all the incoming and outgoing e-mails and I can see that in the initial correspondence, 3 products (ab47535, ab71732 and ab85454) were reported to be faulty.

Could you please update Kate or myself how the other antibodies perform after following some recommendations/tips made by my colleague?

Would you like to get alterative products for ab85454 and ab71732 - but not ab47535?

I look forward to hearing from you and solve this issue as soon as possible.

wetest all the suggestions you gave us, but unfortunately we were unable to get better results in two of the three antibodies we have.

So, for PKN1 (ab47535), we detect specific labelling after the use of your suggestions, although I should remind you that Abcam recommend a dilution of 1/1000 – 1/5000 for IHC-P (see datasheet) and we needed to use 1/500 to get the right signal.

The remaining two antibodies, KDM4C (ab85454)and USP22 (ab71732)still have unspecific labelling and/or no detectable signal even with the suggestions you made.

We don't know if you have any other solution to give us, or if we should apply for the refund.

Best regards,

ANSWER:

Thank you for your response.

My colleague is out of office and she has asked me to look after her customers. I could arrange a credit note for you which you can use in the future.

Please do let me know how you wish to proceed with your enquiry. I look forward to hearing from you soon.

Thank you for your fast reply.

Answering your questions:

1. Please confirm the order numbers (preferably the Abcam order reference number) and date of purchase.
Date of purchase: 03/10/2011
Date of arrival:10/11/2011

2. I would appreciate if you are able to provide information on the blocking steps.

3. Secondary antibodies working well with other antibodies? I would appreciate if you are able to provide more detailed information regarding the secondary antibodies that have been used.

Regarding both questions, our assays were performed using NovoLink™ Polymer Detection System according to the manufacturer's instructions, so we did not use any secondary antibody.

Bear in mind that all our assays were performed in human tissues.

Best regards,

ANSWER:

Thank you for providing this further information.

I appreciate your cooperation and understand your concerns. It is regrettable the results have not been successful.

In order to help with our investigation of this case before deciding how to proceed I would appreciate if you are able to provide some further details. This information will also be helpful to our quality monitoring. I can recommend to the following protocol tips would be beneficial to help optimize the results. I would appreciate if you could you confirm if these have been tried?

1. I can recommend to consider including a permeabilisation step as these are nuclear and cytoplasmic proteins. Try permeabilising in 0.2% Triton for 10 minutes.

2. I can suggest to include 0.2% Tween (a more gentledetergent) in the antibody dilution buffer and wash buffer if you have not already done so. This will help to keep the antibody solubilised and the cells permeabilized.

I will be pleased to provide a free of charge replacement or credit note if these antibodies are not working with ths suggestions provided.

Thank you for your time. I look forward to hearing from you with the requested information and hope we can resolve this case as soon as possible.