OUTLINE
Platelet-associated Ig (PAIg) could be eluted from the platelets. As a result of this procedure platelets get destroyed and the eluted Ig is formed of platelet surface-bound Ig and , mainly, of internal pools of Ig (from a-granules). That is why the caution must be taken when interpreting results.
PROTOCOL
I. GLYCINE-HCl Elution
- Resuspend the platelet pellet in 0,1 mol/L glycine HCl (pH=2.5) to a final concentration 2-10 x 10^8 Platelets/mL
- Incubate at RT for 15 min.
- Centrifuge at 14000 rpm, 5min , RT or 10ѓC
- Neutralize with 1mol/L Tris-base (pH=9.0)
- Dialyze ON, 4ѓC, against PBS
- Centrifuge at 14000 rpm, 5min , RT or 10ѓC
II. ETHER Elution
- Resuspend the platelet pellet (10^8 Platelets/mL) in 400µl [PBS-EDTA 5mM-BSA 2%]
- add 800µl of diethylether
- vigorously mix for 3 minutes
- incubated afterwards at 37ѓC for 45 minutes in 1,5 ml plastic tube without additional shaking.
- let the tube overnight at 4ѓC for ether evaporation
- centrifuged at room temperature for 10 minutes at 5000g
SOLUTIONS
I.
- 0,1 mol/L glycine HCl (pH=2.5)
- 1mol/L Tris-base (pH=9.0)
- PBS
II.
- PBS-EDTA 5mM-BSA 2%
- diethylether extra-pure
ADDITIONAL INFO
If the fraction you are interested in is IgM don't freeze the sample. To avoid the contamination in the tube add NaN 3 to the final concentration of 0,05%
REFERENCES
- Bowditch, R.D., Tani, P., Fong, K.C. and McMillan, R. (1996). Characterization of autoantigenic epitopes on platelet glycoprotein IIb/IIIa using random peptide libraries. Blood 88(12): 4579-84
- Kosugi, S., Tomiyama, Y., Shiraga, M., Kashiwagi, H., Mizutani, H., Kanakura, Y., Kurata, Y. and Matsuzawa, Y. (1996). Platelet-associated anti-glycoprotein (GP) IIb-IIIa autoantibodies in chronic immune thrombocytopenic purpura mainly recognize cation-dependent conformations: comparison with the epitopes of serum autoantibodies. Thromb Haemost 75(2): 339-45.
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