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| == SDS-PAGE Westerns ==
| | #redirect [[Protein blot (Western) hub]] |
| === Overview ===
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| A Western Blot allows for the semiquantitative determination of protein expression.
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| 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.
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| === Materials ===
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| *2x PLS
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| *PBS-T
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| *5% milk in PBS-T
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| ===Procedure===
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| ====Prepare Samples====
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| *Boil Samples for 5 min, cool on ice 1 min, spin 1 min
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| *Ladders: 8ul ladder + 8ul 2x PLS
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| **High mol weight 30KDa - 220 KDa
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| **Low mol weight 6.5KDa - 45 Kda
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