Dear Doustimotlagh

I think the differential centrifugation is the best. Kindly see the following links.

Regards

http://eu.montana.edu/BioScience/documents/Meta_blood_processing_procedure.pdf

http://stevegallik.org/pdf/CellBiologyOLM_Exercise05_2012.pdf

Please refer to the attached PDF. This method works best in our hands

Thank you so much for your response.

The methods were perfect for isolation of erythrocyte membranes, but they had few differences, for example in the first paper; you used 10000 rpm and in the last paper you used 200000 rpm for centrifugation. unfortunately, we do not have a centrifuge  with 200000 rpm. Are these methods have similar efficiency?

You can centrifuge at 10000 rpm for 15 min. The idea is to get clean hemoglobin- free membranes. 20000 rpm will help in quicker sedimentation and slightly improved yield. Otherwise, the membranes will have the same enzymatic properties and make-upeven if you sediment at 10000rpm for 15 min..

Thanks a lot.

Hi Surendra , regarding your comment, how do you avoid hemoglobin clumping on your membranes, what precautions are critical in this respect? Thank you

If you wash repeatedly pellet with 20 mM Tris , you will get ghosts practically free of Hb. For eg. see the attached PDF and original reference therein.