Anti-Renalase 抗体 (ab31291)

Thanks for your reply.

I think it will be very important, based on my knowledge of the situation, to get some specific details from the authors of the paper whose experiment you wish to reproduce. I suggest sending another email within a day or 2 if you do not hear from them.

I look forward to hearing how things go.

Regarding your enquiry, I have spoken with my colleague John who you spoke with on August 9. He mentioned advising you to contact the authors of the study you are attempting to duplicate. I just wanted to know if you have heard from them yet

Thanks in advance for the additional information.

Thank you for your email.

Yes, with the peptide you can make the standard curve.

When using unknown samples, you would just coat the well with the samples, but not the peptide. The peptide would be in separate wells. Also, try several amounts of thesample as to determine which will give you a good signal.So, either peptide or sample are coated and then you would use the antibody to detect peptide or sample. Subsequently, a secondary antibody (HRP or AP conjugated) will be used to obtain the signal/read out.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Thank you for contacting us.

ELISA assay is used for determining the activity of the antibody by applying serially diluted antibody to micro-wells that are coated with the immunizing peptide. Please find below the protocol that was used, including the peptide amount:

Wells are coated with the immunizing peptide and serially diluted antibodies are applied and measured.



Coat 0.1ug peptide in 100ul PBS per well on high binding ELISA plates overnight at 4C.
Wash 3 times with 300ul PBS per well.
Block with PBS/0.1%(v/v) Tween-20 (PBST) containing 3%(w/v) skimmed milk (blocking buffer) for 1h at 37C
Wash 3 times with PBST
Make serially dilutions of antibody in blocking buffer and apply 100ul per well.
Incubate 1h at 37C with the plates covered
Wash three times with 300ul PBST
Apply secondary antibody (AP-conjugate) at appropriate dilution in blocking buffer, 100ul per well
Wash four times in 300ul PBST
Wash 2 times in 300ul PBS
Add 50ul per well p-nitro phenyl phosphate (pNPP) from Sigma and incubate for 30min at 30C
Stop the reaction with 50ul 1M NaOH.
Read at 405nm



Phosphate buffered saline (PBS): 20mM Sodium phosphate, pH7.4 in 150mM NaCl.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Thank you for your enquiry. Unfortunately we do not provide the standard protein. This product was tested in peptide-ELISA, with the immunizing peptide bound to the plate. I am sorry I can't be of more assistance in this occasion. If there is anything else that I can help you with, please do not hesitate to contact me.

Thank you for your enquiry. I am sorry to hear that you are experiencing difficulties with this product ab31291 in western blot. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 120 days of purchase), and if it appears that the antibody is at fault, a replacement/credit note/refund will be offered. After looking at the protocols you used, I am surprised that even 95ug of protein, you still did not manage to get any bands. However, it is difficult for me to determine the cause without more details of your WB protocol. I would therefore appreciate if you can please clarify the following items. Most customers who use milk as a blocking agent tend to have problems detecting bands. At Abcam, almost all of our products have been tested with 5% BSA. Sometimes, a different blocking buffer might cause cross reaction between the blocking agent and antibodies, therefore causing weak bands or no bands at all. I would recommend trying 5% BSA, if you have not already done so. Also, cross reaction between the antibodies with the diluent could also cause the lack of signals. We would normally use 1% BSA to dilute the primary and secondary antibodies. You can also use PBST only. Can you confirm that the second antibody you used also managed to produce good results with other primary antibodies? In some cases, the problem may be with the secondary antibody not working. Please try incubating the primary antibody overnight at 4oC. Incubation at this temperature and for longer duration will ensure proper and sufficient binding to the protein of interest. Could the no signal you are experiencing be due to poor transfer of protein to membrane? Did you check the transfer efficiency? This can be simply done with a reversible stain such as Ponceau S. I hope the above recommendations may already help you. If you have already tried the above suggestions and still experience problems, please do not hesitate to contact me with details of your order (purchase order number, date of purchase, shipping address/purchasing agent information). Also, please advice me on how you would like to proceed with your enquiry, so that I can immediately arrange for a replacement or refund to you.

Thank you for your enquiry. I have copied the ELISA protocol below: Peptide ELISA - Plates coated with immunising peptide at 1ug/ml, 4C overnight - Plates blocked blocked in 2.5% skimmed milk in TBS-T (TBS + 0.05%Tween-20) for 1 hr at room temperature - Antibody diluted 1/1000 in blocking buffer, 1hr 37oC - Secondary antibody, Anti-goat Alkaline Phoshatase - Readout using p-nitrophenyl phosphate. I am sorry but the product was not tested on plasma from human or mouse but it has　been tested by western blot on human and mouse kidney. I hope this information will be helpful. Have a nice day.