Anti-Lrp2 / Megalin抗体(ab56014)

Thank you for contacting us. I did BLAST searches of our LRP2 antibodies against C.elegans and found that the highest match was with ab56014 with 78%. Usually we like to say that 85% homology between the immunogen and the species will give likely reactivity. This antibody also had a 75% homology with LRP1, so this may not be a good choice for you. All other LRP2 antibodies had lower homology with C. elegans and are not likely to react.

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.

We do not have set expiration dates for our products. Most antibodies are stable and can last for anywhere from a few months to several years if stored properly, so we strongly recommend that you follow the storage instructions on the datasheet for the antibody you purchased. These conditions will vary among our antibodies, therefore, it is important to verify the storage conditions for each of our products when you receive them. We guarantee all of our products to work for at least 180 days from the date of purchase when stored correctly.

Storage at +4C for this long of a period may or may not have affected the product. I would recommend use as normal to determine if there has been any adverse affect.

For more information on antibody storage and stability, please visit our Antibody Storage Guide on our Protocols and Troubleshooting Tips page (https://www.abcam.com/index.html?pageconfig=popular_protocols). I have included this as an attached pdf as well.

I hope this information helps. Please do not hesitate to contact us if you need anything further.

Thank you for providing that extra information.

The band that can be expected with this lysate is the predicted 517 kDa, however, to our knowledge, this lysate has not been specifically probed for Lrp2 and I cannot therefore be sure of this. Additionally, ab56014 has not been used to detect the endogenously expressed protein in western blotting in house (which is also seems to betrue of the Sc-25470). Only recombinantly expressed protein fragments have been detected. This makes performing the western blot considerably easier as the transfer of the smaller recombinant protein is much less problematic than transferring a 517 kDa protein.

Can I ask, are you seeing any bands after performing the western blot or is the membrane completely blank? If you could possibly share an image of the blot obtained that would be very helpful. What was the result of the "no primary" control?

I would advise tryingthe following in order to improve the resultsobserved to date:

1.I would prepare the protein samples by heating for 10 minutes to 70 degrees. If you are not already doing so I would also used a reducing agent in the loading buffer. This is not necessary with the control lysate as this is already included (butthe heating step should still be performed).

2. In order to improve the transfer and binding of the protein to the membrane I would use PVDF membrane and make sure to pretreat in methanol for 1-2 minutes, followed by incubation in ice cold tranfer buffer for 5 minutes.

3. I would use a very low percentage gel in order to improve the transfer, as well as adding 0.1% SDS to the tranfer buffer as this prevents the precipitation of the protein in the gel. I would also remove the methanol from the transfer buffer as this tends to remove the SDS. Methanol is only necessary in the transfer buffer if using a nitrocellulose membrane. Removing the methanol also promotes the swelling of the membrane which allows large proteins to transfer more easily.

I hope these suggestions improve the results observed so far. Performing a western blot with such a large protein will be quite difficult and may require quite a lot of optimisation in order to get a good result.

I look forward to receiving yourreply.

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to clarify a few details in order to be able to understand the situation more fully and hopefully be able to provide some suggestions in how to improve the results currently observed.

1. could you share with me what the other anti-Lrp2 antibody used was (manufacturers name and catalogue number)?

2. what lysis buffer was used for your other samples?

3. what transfer buffers were used (composition)?

4. how long was the transfer performed for and at what voltage?

5. you mentioned that the transfer was verified by staining of the membrane, do I assume right that the 500-600 kDa region transferred ok? Could you share the images of theseresults with me?

6. how long was the membraneexposed for?

7. has a "no primary" control been performed?

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

Thank you for contacting us and reporting the problems observed with using anti-Lrp2antibody ab56014 with Kidney (Human) Tissue Lysate (ab44687).

In order to understand the problems your customer is experiencing further could you please pass on the questionnaire attached to this email for them to fill out. Performing western blotting with such a large protein can be very tricky and fully understanding the protocol your customer has used may shed further light on what may be the problem.


I look forward to receiving your reply.