20150226 WB KIR4.1

REAGENTS:
– Tris 3M pH 8.8
– Tris 2M pH 6.8
– Acrylamide 40%
– Temed
– PSA 10%
– SDS 20%
– B-mercaptoetanol
– Isopropanol
– Blocking buffer (Licor)
– Primary Ab: aKir4.1 1/5000 (Millipore AB8518)
– Secondary Ab: GAR800 1/7500
– Equipment for WB gels
– Equipment for WB blotting

ASSAY:
1. SDS-page gel 10% resolution gel and a 4% concentracion gel. 1h gelification aprox.
*Resolution:
Tris pH 8.8 3M: 1.25ml
Acrilamide 40%: 2.50ml
H2O: 6.15ml
SDS 20%: 50ul
PSA 10%: 100ul
Temed: 10ul
*Concentracion:
Tris pH 6.8 3M: 0.75 ml
Acrilamide 40%: 1 ml
H2O: 8.25 ml
SDS 20%: 50 ul
PSA 10%: 100 ul
Temed: 10 ul
2. Prepare the samples doing a dilution 1/2 with laemly. Load 30ul per well, put it at 95ºC for 5 minutes and do a centrifuge 13,2rpm 1 minute.
3. Load samples (30ul)
4. Run with running buffer 3-4 hours. at 20 – 40mA
5. Transfer to a nitrocellulose membrane o/n 30V 4ºC
5. Blotting: wash the membrane with water and cut it
6. Blocking: blocking buffer – PBS 1:1 1h
7. Primary antibody (aKir4.1) 1/5000 2h
8. Wash PBS-tween20 0.1% x3 5′
9. Secondary antibody GAR800 1/7500
10. Wash PBS-tween20 0.1% x3 5′
11. Wash PBS x1 5′
12. Read with Odyssey equipment

RESULTS:

CONCLUSIONS:
We must confirm the presence of oligomeric kir4.1 with low glycosidation in elution 1, 2 and 3, but we also see kir4.1 in the flow-trough (FT).
We must refine the experimental assay.