Western Blots: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Line 1: | Line 1: | ||
== SDS-PAGE Westerns == | == SDS-PAGE Westerns == | ||
=== Overview === | |||
A Western Blot allows for the semiquantitative determination of protein expression. | |||
Crude cell lysates are loaded into a polyacrylamide gel containing a denaturing agent which give all the proteins a net negative charge. A current can then be passed through the gel and the proteins will migrate through the gel, with the largest proteins traveling the slowest, resulting in a lane where proteins become separated on the basis of their weight. The proteins can then be transferred from the gel onto a membrane (often nitrocellulose or PVDF), which can then by incubated with an antibody directed against the protein of interest. By using a detector conjugated to an antibody one can now detect specifically the protein of interest. | |||
=== Materials === | === Materials === |
Revision as of 10:32, 31 August 2006
SDS-PAGE Westerns
Overview
A Western Blot allows for the semiquantitative determination of protein expression. Crude cell lysates are loaded into a polyacrylamide gel containing a denaturing agent which give all the proteins a net negative charge. A current can then be passed through the gel and the proteins will migrate through the gel, with the largest proteins traveling the slowest, resulting in a lane where proteins become separated on the basis of their weight. The proteins can then be transferred from the gel onto a membrane (often nitrocellulose or PVDF), which can then by incubated with an antibody directed against the protein of interest. By using a detector conjugated to an antibody one can now detect specifically the protein of interest.
Materials
- 2x PLS
- PBS-T
- 5% milk in PBS-T
Procedure
Prepare Samples
- Boil Samples for 5 min, cool on ice 1 min, spin 1 min
- Ladders: 8ul ladder + 8ul 2x PLS
- High mol weight 30KDa - 220 KDa
- Low mol weight 6.5KDa - 45 Kda