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Summary: 1
Alkylation of SDS-PAGE Samples
General Method
1. Dissolve protein sample in freshly prepared 100 mM Tris-HCl, pH 8, 2% SDS, 50 mM DTT, 20%
glycerol. The pH should be 7.5 - 8.5 for alkylation.
For immunoprecipitations, remove as much volume as possible from Sepharose beads after final
dH2O wash (use gel loading tip). Estimate volume of remaining beads (~10-20 µl). Add equal
volume of 100 mM Tris-HCl, pH 8, 2% SDS, 50 mM DTT, 20% glycerol.
2. Incubate at 85°C for 10 minutes.
3. Add exactly 1/5 volume of freshly made 1M iodoacetamide. Vortex. Spin briefly.
1M iodoacetamide = 0.1 g iodoacetamide + 450 µl dH2O. Aim for 2X molar excess over DTT.
For immunoprecipitations, the brief spin will pellet Sepharose beads.
4. Incubate at room temperature for 15 minutes.
5. Load gel.
Antibodies Manual Method for Immunoprecipitations
1. After immunoprecipitation, remove as much volume as possible from Sepharose beads after final
dH2O wash (use gel loading tip). Assume that 10 µl of volume remains (~10-15 µl of beads).
2. Add 10 µl of 2% SDS + 20 mM DTT.
3. Incubate at 85°C for 10 minutes. Spin for 2 minutes at top speed in microfuge to pellet Sepharose
beads. Transfer as much SN as possible to fresh tube (use gel loading tip).
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