User:Karmella Haynes/Notebook/PcTF Genomics/2015/08/12: Difference between revisions
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# Gal4EED-luc: H380_MV11 + g034_pSPgRNA | # Gal4EED-luc: H380_MV11 + g034_pSPgRNA | ||
# Gal4EED-luc: H380_MV11 (no gRNA) | # Gal4EED-luc: H380_MV11 (no gRNA) | ||
'''SDS-PAGE''' | '''SDS-PAGE''' | ||
* Protocol: http://openwetware.org/wiki/Haynes:PAGE | * Protocol: http://openwetware.org/wiki/Haynes:PAGE | ||
* Gel: Nupage 4-12% Bis-Tris | * Gel: Nupage 4-12% Bis-Tris (12 wells) | ||
* Buffer: NuPAGE® MOPS SDS | * Buffer: NuPAGE® MOPS SDS | ||
* Chamber: Invitrogen XCell SureLock | * Chamber: Invitrogen XCell SureLock | ||
* Protein standard: | |||
* Protein sample prep: | * Protein sample prep: | ||
** Final volume = 20 μL | |||
** Sample buffer: | |||
Add protein samples to 1.5 mL tubes. Dilute the stock proteins in an appropriate buffer. Make sure the protein sample volume = the final volume - (concentrated sample buffer volume + 1.0 μL 1M DTT) | |||
Example 1: 20 μL final loading volume - (5.0 μL 4x sample buffer + 1.0 μL 1M DTT) = 14.0 μL protein | |||
Example 1: 20 μL final loading volume - (10.0 μL 2x sample buffer + 1.0 μL 1M DTT) = 9.0 μL protein | |||
Calculate the number of lanes used = protein samples + protein standard(s). If this is less than the total number of lanes, prepare "dummy" samples (sample buffer + DTT + water) to fill empty wells | |||
Heat samples, excluding the protein standard(s), @ 100°C/ 5 min. Cool at room temp. | |||
Revision as of 16:01, 12 August 2015
 Pc-TF Genomics
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08/12/15
Castone Western - gel and transfers
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