Anti-KDM4A / JHDM3A / JMJD2A 抗体 (ab24545)

I'm sorry to hear that ab24545 did not work for you in western blot.

As you requested, I have set up an Order 1217875 for ab70786 free of charge. The only vial in stock is being transported to the US, and the expected delivery date to you is ***

Please do not hesitate to contact us with any further questions.

Thank you for your response and detailed protocol description.

Did you try lowering the exposure time of the film until the background luminescence was gone? Also, please respond if you would like a replacement vial of the same antibody, a different antibody, or a refund.

I look forward to your reply, your information will be added to our internal notes and is greatly appreciated.

Thank you for contacting Abcam.

Based on the image sent for ab24545, there is definitely non-specific binding occurring. In order to block some of the non-specific binding, please attempt the troubleshooting steps listed below, so we can determine whether the antibody is properly recognizing its immunogen. If you have already performed these troubleshooting steps, please reply with details regarding your sample, protocol and troubleshooting steps that have been taken. These will not only help me to fully understand the problem but are essential should further testing of this product be needed as well. Could you also provide the Abcam order confirmation number or the PO and date of purchase for this product?

Blocking of non-specific binding might be absent or insufficient.
Increase the blocking incubation period and consider changing blocking agent. Abcam recommends 5% non-fat dry milk, 3% BSA, or normal serum for 30 min. These can be included in the antibody buffers as well.

The primary antibody concentration may be too high.
Titrate the antibody to the optimal concentration, incubate for longer but in more dilute antibody (a slow but targeted binding is best).

Incubation temperature may be too high.
Incubate blot at 4°C.

The secondary antibody may be binding non-specifically or reacting with the blocking reagent.
Run a secondary control without primary antibody. This is probably not the issue since I'm assuming you used the same secondary antibody for ab17721 which worked well.

Cross-reaction between blocking agent and primary or secondary.
Add a mild detergent such as Tween20 to the incubation and washing buffer (phospho-specific protein). Milk contains casein which is a phosphoprotein; this is why it causes high background because the phospho-specific antibody detects the casein present in the milk. Use BSA as a blocking reagent instead of milk.

Washing of unbound antibodies may be insufficient.
You have already attempted this troubleshooting step which did not solve the problem.

Your choice of membrane may give high background.
Your background for the other antibody looks fine, so it should not be the membrane.

The membrane has dried out.
Probably not the case since your other antibody staining was clean.

If these troubleshooting steps do not solve the problem we will refund or replace your product as stated in our AbPromise guarantee if the antibody is being used in the applications / species listed on the datasheet.

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

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link:
https://www.abcam.com/index.html?pageconfig=resource&rid=15447

Thank you for your enquiry. I am sorry to hear that you have been experiencing problems with ab24545 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 free replacement/credit note/refund will be offered. I can confirm that this product will work well in human samples. However, it has yet to be tested in mouse samples and we can not guarantee you will obtain good results. I have looked through the protocols you used and have a few questions and suggestions that might help you resolve the problem. - One of the most common cause of non-specific bands is there is an over loading of protein. We recommend using 20-40ug per well. This will eliminate the non-specific bands. One other reason is that the protein target may form multimers. Try boiling in SDS-Page for 10 minutes rather than 5 minutes to disrupt multimers. - The target in your protein sample might have been digested (more likely if the bands are of lower molecular weight). Please make sure that you incorporate sufficient and fresh protease inhibitors in your sample buffer. - As this antibody is supposed to detect a band of approximately 120-130kDa, using an 8% gel is recommended so that the proper molecular weight proteins can be separated accordingly. - At Abcam, almost all of our products has 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. - Can you confirm that the second antibody you used also managed to produce good results with other primary antibodies? Could it be causing the weak signal? Are the non-specific bands weak or strong? If the non-specific bands are stronger, then the protein of interest is not abundantly present in the cell extract. - How much Tween was in the TBST solution? In general, a 0.05% TBST would be sufficient as a washing buffer. Also, was the washing done 15min x 3 times? This is because over washing will eventually cause you to lose signals and insufficient washes will not be able to rid the non-specific bands. I am sorry for the string of questions but 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 the answers to the above (including an image), and details of your order (contact information, shipping address/purchasing agent, etc.). 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 for you.