I am going to measure Glut1 in erythrocyte membrane using ELISA assay. Checking the literature I have found different protocolos for collecting membrane ghosts. Can you suggest a particular one?
You might want to consider using the KB (Kleihauer Betke) stain to treat the RBCs. This acid stain would make adult intercellular hemoglobin soluble and leak out from the RBC, and thus make RBC become ghost like.
I got this idea from the experience of using KB acid stain kit to differentiate the fetal RBCs (stain homogeneous pink on the smear) from the adult RBC (ghost like post the KB acid stain on the smear) when performing the KB testing.
I use the classical method described in many papers in the literature, with some variations. For instance you can see the protocol in
Minetti G, Ciana A, Profumo A, et al. Cell age-related monovalent cations content and density changes in stored human erythrocytes, Biochim Biophys Acta 1527 (2001) 149-55.
or
Marchesi VT, Palade GE. The localization of Mg-Na-K-activated adenosine triphosphatase on red cell ghost membranes, J Cell Biol. 35 (1967) 385-404.
It is a procedure which requires a few hours, but ensures a good purification of ghosts.
This is the method we use in our laboratory.Very simple and fast,suitable to study all membrane proteins.
See https://www.researchgate.net/post/How_can_I_produce_resealed_erythrocytes_ghost_in_the_best_and_fastest_way
Hello Simona Giardina,
There are lots of protocol to prepare RBC membrane ghosts, but i'm suggesting you one which been used in our laboratory and published. If you need whole article, just knock at once.
2.8. Preparation of Erythrocyte Ghosts
To erythrocyte pellets (30 x 106 cells), 25 mL of NaH2PO4 was added (10 mM NaH2PO4; pH was adjusted to 7.4 by 1N NaOH).The suspension was centrifuged at 20,000 g for 40 minutes. The process was repeated 3 to 4 times until the supernatant was colorless or a faint pink. The pellet was again washed in PBS, and erythrocyte ghosts were suspended in CM.
Best regards,
Babu
https://www.ncbi.nlm.nih.gov/pubmed/10846827?dopt=AbstractPlus
http://eu.montana.edu/BioScience/documents/Meta_blood_processing_procedure.pdf
Simona Giardina
hi,
I designed a novel peptide that is an inhibitor of acetylcholinestrase.
there is this enzyme on the red blood cell membrane, so I need ghost or free hemoglobin RBC to test my peptide.
we tried this method previously and it works:
Al-Jafari, A. A., Kamal, M. A., Greig, N. H., Alhomida, A. S., & Perry, E. R. (1998). Kinetics of human erythrocyte acetylcholinesterase inhibition by a novel derivative of physostigmine: phenserine. Biochemical and biophysical research communications, 248(1), 180-185.
but nowadays I can't find an ultracentrifuge and I'm struggling to start my thesis.
I try this method too but it doesn't work at all:
Schwoch, Gerhild, and Hermann Passow. "Preparation and properties of human erythrocyte ghosts." Molecular and cellular biochemistry 2.2 (1973): 197-218.
the maximum speed of our centrifuge is 20,000 g.
I'm short of time.
PLEASE HELP ME :((
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