Anti-IRAK-1 抗体 (ab238)

I have checked your new order 1217270 and it looks that both antibodies (abhttp://ops.adminsite.com/admin/data/fs_ab.cfm?&intAbID=119289 and abhttp://ops.adminsite.com/admin/data/fs_ab.cfm?&intAbID=135027) are out of stock at the moment. We have reordered the items but it may take a week to get them. According to our record, we should be able to get them by 18th December.

Could you please drop me an e-mail to confirm if you are happy to wait.

I apologize for any inconvenience caused and look forward to hearing from you soon.

Thank you for getting back to me.

This is to confirm that I have placed a new order for you - for one vial of ab238 and another vial of ab64171 as free of charge replacements and the new order number is 1217226.

I would kindly advise you to run a non-stimulated positive control cell line which expresses the target protein endogenously and to apply lysis buffer (i.e. RIPA) which contains a mixture of different proteinase inhibitors to prevent the degradation of the protein of interest.

Recommended positive controls:

ab238 - THP-1 or HeLa whole cell lysate;

ab64171- Jurkat whole cell lysate

I hope the second vials will perform better, and please do let me know how you are getting on with these two products.

Good luck!

Thank you for getting back to me and for passing some useful details. Your co-operation is much appreciated.

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.

I can certainly offer replacement vials to you if you wish to carry on with the experiments.

Could you please confirm the Abcam Order Number or you Purchase Order Number and the date of purchase.

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

According to Swiss-Prot database, IRAK has 4 different isoforms (P51617-1, P51617-2, P51617-3, P51617-4) and these alternative splice variants have different sizes. You can find some further details at this site:

http://www.uniprot.org/uniprot/P51617

One more comment that RT-PCR data should be treated carefully since, there is no clear correlation between mRNA and protein levels and sometimes it is not easy to predict the protein expression levels from quantitative mRNA data.

There are several publications available which delineate the technical boundaries of the approaches for quantitative analysis of protein expression and reveal that simple deduction from mRNA transcript analysis is insufficient.

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

Thank you for getting back to me.

I can find the 3 pdf files attached to your response but it seems that the ppt (Powerpoint) file did not come through.

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

1) Control for siRNA experiments:

Have you done a time-course experiment with siRNA treated cells to select the most optimal time point for the studies?
How have you selected the sequence of siRNA? Have you tried more than one SiRNA sequence? Could you check if the sequence corresponds correctly to IRAK and CARD10 protein?
Have you checked the transfection efficiency and optimized the length of time for knockout to take effect?
Have you checked the transfection by fluorescence tags and the knockout by RT-PCR?
Have you used the correct endogenous positive and negative controls? Have you applied scrambled siRNA or standard negative siRNA controls?



2) RIPA buffer:

Laemmli buffer does not contain any proteinase inhibitors to protect the target protein form degradation. I would suggest using RIPA buffer and you can find the composition of the this solution at this site:

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



3) Positive control:

Since the cells are stimulated with IFN gamma, this may modify/alter the expression pattern of the target. It would be worth running good positive control alongside with the samples. It would be also be worth checking the cell viability after treatment.

ab238 - THP-1 or HeLa whole cell lysate;

ab64171- Jurkat whole cell lysate

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.

I am sorry to hear that you have been experiencing problems using these two products (ab238 and ab64171) in the application that you wish. Thank you very much for sending the WB images.

In order to assess the quality of our products I would ask that you complete a brief questionnaire relating to the application used. Often it is possible to make suggestions that may help resolve problems experienced using a particular product.

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.

All our customer feedback, including complaints are monitored weekly by our in house technical support team. If a product is at fault the technical support team will consider removing the product from our catalogue in order to avoid future customer inconvenience.

Could you please provide some further details of the protocol used and complete the following form (attached as a word document).

I am particularly interested in the following information:

- lysis buffer used,

- positive control,

- summary of the siRNA experiment (sequence for siRNA, transfection efficiency, endogenous positive and negative control)

Thank you for your understanding and co-operation in this matter. I look forward to hearing from you soon and resolving this issue as soon as possible.

Thank you for your inquiry.

I can confirm that the protocol for flow cytometry that we know of for this antibody includes an incubation time of 16 hours at 4C.

Please feel free to use your standard protocol if you find that this is working too for this antibody. In case the staining is week I can suggest too prolong the incubation time. A longer incubation will achieve an optimal saturation.

If in your hand a shorter incubation time is working well, i would like to encourage you to also write an Abreview. Other researchers will value this information. And you would earn Abpoints that can be redeemed as discount on future purchases or as Amazon vouchers.

I hope this information is helpful and wish you good luck with your research.

I am sorry to hear that the result did not improve.

To proceed further in this case could you let me know, how the antibody was stored and what was the purchase order number?

Thank very much for your cooperation in this case!

Thank you for submitting an Abreview of ab238. As you may have noticed, your review has now been published on our website.

Since you obtained poor results using the antibody in a tested species and application, we would like to follow up on this to see if we can possibly improve the results you are seeing with this antibody.

Based on the information in your review, I feel that altering the step in protocol may help toimprove your results. Did you try any troubleshooting of protocol?

Additionally, this antibody is covered by our Abpromise, so if the antibody was purchased within the past 6 months, I would be happy to discuss a refund or replacement with you to resolve this issue.

I look forward to your reply.