Anti-Mannose 6 Phosphate Receptor (Cation independent) antibody [2G11] - Late Endosome Marker (ab2733)

Brief protocol:



1. 50,000 - 150,000 Cells lysed in 20uL running buffer containing mercaptoethanol for 5min in 95C heat block (no protease inhibitor used)



2. Samples (20uL) run on gel with protein ladder for approx 90 mins.



3. Transferred for 2hr - very good transfer of protein marker seen



4. Blocked for 60 mins at RT with 5% milk



5. Incubated with primary antibody (1 in 2000 dilution) overnight at 4C



6. Incubated with secondary antibody for 1hr at RT



7. ECL applied for 1 min



8. Membrane developed (images below).

ANSWER:

Thank you, I received the data this time. For a protocol modification, I suggest trying preparing a sample by heating at 60C for 10 minutes instead of 95 for 5 minutes to minimize aggregation of the trans-membrane domains. It is strange that both antibodies give you this result, and I still think there may be some issue with the sample entering the gel and/or transferring effectively, even though the standards do.

Another modification is to remove methanol from the transfer buffer and add SDS to a final concentration of 0.1%, to encourage transfer of this large protein.

Are you confident that the secondary antibody is not reacting with the samples? Is the secondary specific for a subclass of mouse IgG?

These modifications are unlikley to remove the band at 70 kDa. We do have other antibodies against the receptor, so if these modifications fail, I can send a vial of something else to try, such as ab32815 or ab124767.

I have been having trouble with two Abcam IGF2R (CI-M6PR) antibodies, a b2733 and ab8093, which I have been attempting to use for Western Blotting.

Both antibodies have failed to show a band in the expected region (approx 250Kd) but have instead shown a band at around 70Kd. I have confirmed the cell lines that I am using (CMK and K562) express IGF2R using PCR and flow cytometry. I have attached a document containing the gels run for the two proteins. I have tried each antibody 3 times now with similar results. I have run secondary only controls, which do not show this band.

Do you have any suggestions on how to improve my results?

ANSWER:

Thank you for bringing this to our attention. I will need a few more details of your protocol. I was not able to open the file you sent.

Can you please tell me how you prepared your lysates (did you use protease inhibitors), and how you heated the samples to denature. I am concerned that the receptor may have aggregated upon boiling and did not enter the gel effectively, or did not transfer to the blot well. Did you check for efficacy of transfer with a Ponceau Red stain or equivalent?

How much sample did you load per lane of the gel? How did you block the membrane? What concentration of antibody did you apply to the membrane and how long did you incubate with the antibody?

I am using this antibody in WB, but am unable to detect any bands.

ANSWER:

Thank you for contacting us regarding ab2733.

I have confirmed with the laboratory that this particular antibody is only batch tested in IF, but has been tested in WB. I have attached an image for you.

We have recently done some optimisation with TA gels (and 0.45 um membranes) and we found using 10% methanol +0.04% SDS or 10% ethanol +0.04% SDS for the transfer buffer worked really well. We also found that it was best not to heat the lysates to 100C for TA gels (no heating was best but 60C was ok). We haven’t tried this procedure with this ab though so I can’t say if it will help solve your problem for sure.

The presence of SDS increases solubility of membrane proteins and high MW proteins allowing efficient elution from the gel.

As standard we denature at 95C. Reduced denaturation temperature (60-70C) or no denaturation is often recommended for membrane proteins/high MW proteins to reduce precipitation and failure of the protein to migrate onto the gel during electrophoresis.

I hope this information is helpful. Please do not hesitate to contact us if you have any additional questions or if your results do not improve with this antibody.

Thanks, I have just tried with BSA as blocking agent and I did have more success but with also more a specific bands from ms9-II cells that overexpress M6PR. I will also try ab32815 to see if the later ab shows less a specific bands with my ms9-II cell extract.

Thanks again and you have been really very helpful

ANSWER:

I'm glad that you have been seeing better results with blocking with BSA. If you are now having difficulty with non-specific bands, increasing the dilution of the primary and secondary antibody as well as adding 1 % BSA and 0.2% Tween 20 into the diluents may help.

If you would like me to have a look at your protocol to see if there is any other advice I can think of then please do fill in the form and I'd be happy to try and help. Otherwise, I wish you all the best with your experiments.

Thanks for your advice. If possible I would prefer to try the alternative antibody you suggest (ab8093). Would you be able to send me a replacement vial?  

ANSWER:

Thank you for your reply.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement for ab8093.

To check the status of the order please contact our Customer Service team and reference this number. Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.