Anti-Lrp2 / Megalin抗体(ab101011)

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 your enquiry regarding ab101011.

The peptides are selected from the cytoplasmic domain: topological domain: 4447 – 4655.

I hope this helps and if I can assist further, please do not hesitate to contact me.