Chemicals and solutions for the IEF of mitochondrial proteins
Sample solution A: 5M urea, 2M thiourea, 2 CHAPS, 2 NDSB-256, 2mM tributylphosphine, 0.5 IPG buffer.
Prepare the solution without TBP and IPG buffer, filter through a 0.2-0.44 m filter, aliquot and store at -20° C. TBP and IPG buffer should be added to the sample solution only prior the use!
Sample solution B: 7M urea, 2M thiourea, 4 CHAPS, 1 ASB-14, 2mM tributylphosphine, 0.5 IPG buffer.
Prepare the solution without TBP and IPG buffer, filter through a 0.2-0.44 m filter, aliquot and store at -20° C. TBP and IPG buffer should be added to the sample solution only prior the use!
Equilibration buffer: 6M urea, 50mM Tris-HCl, 2 SDS, 30 glycerol, 5mM tributylphosphine, pH 6.8.
Filter the solution through a 0.2-0.44 m filter, aliquot and store at -20° C.
Urea; MW=60.06, 99.5; Pharmacia Biotech, No. 17-1319-01
60.36 g/mol - 100
5 M – 3.018 g/10 ml
6 M – 6.6216 g/10 ml
7 M – 4.2252 g/10 ml
Thiourea; MW=76.12; Sigma, T-7875
2 M – 1.5224 g/10 ml
CHAPS; MW=614.9, 98+; Sigma, C-5070
2 (w/v) - 200mg/10ml
4 (w/v) - 400mg/10ml
NDSB-256; MW=257.4, >99; Calbiochem, No. 480010
2 (w/v) – 200 mg/10 ml
ASB-14; MW=434.7, >99; Calbiochem, No. 182750
1 (w/v) – 100 mg/10 ml
Tris 7-9; MW=121.1, 99; Sigma, T-1378
50 mM - 0.06116 g/10 ml
Sodium dodecyl sulphate (SDS); MW=288.38, 99.0; BDH, No. 44244
2 (w/v) – 200 mg/10 ml
Glycerol, MW=92.09, 99.9; Sigma, G-7893
30 (v/v) – 3 ml/10 ml
TBP; MW=202.32, 95; Fluka, No. 90827
2 mM – 0.7 microl/350 microl
5 mM – 50 microl/10 ml
IPG buffer, pH 3-10NL; Pharmacia Biotech, No. 17-6000-88
0.5 (v/v) – 5 microl/1000 microl (1.75 microl/350 microl)
2004 02 11 – Ove Julius