|Product Includes||Quantity (with Count)||Storage Temp|
|GST-Human PAK1-PBD||1 x 600 µg||-20°C|
|Rac1 Mouse Antibody||1 x 50 µl||-20°C|
|GDP||1 x 50 µl||-80°C|
|GTP γS||1 x 50 µl||-80°C|
|Glutathione Resin||1 x 3 ml||+4°C|
|SDS Sample Buffer||1 x 1.5 ml||+4°C|
|Lysis/Binding/Wash Buffer||1 x 100 ml||+4°C|
|Spin Cup and Collection Tubes||1 x 30 ea||RT|
GTPγS: Store at -80°C
GDP: Store at -80°C
GST-Human PAK1-PBD: Store at -20°C
Rac1 Mouse mAb: Store at -20°C
Lysis/Binding/Wash Buffer: Store at 4°C
Glutathione Resin: Store at 4°C
SDS Sample Buffer: Store at 4°C
Spin Cup and Collection Tubes: Store at 4°C
NOTE: We recommend making 1 mg/ml lysate in 1X Lysis/Binding/Wash Buffer for the following steps.
Perform the following treatments, GTPγS (positive control) and GDP (negative control), to ensure the immunoprecipitation procedures are working properly. Use 500 µg of cell lysate for each treatment. For best results, aliquot GTPγS and GDP at first use to minimize freeze/thaw cycles.
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
NOTE: CST recommends loading prestained molecular weight markers (#74124, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights. 4-20% acrylamide or Tris-glycine gel provides best separation.
NOTE: LumiGLO® substrate can be further diluted if signal response is too fast.
NOTE: Due to the kinetics of the detection reaction, signal is most intense immediately following LumiGLO® incubation and declines over the following 2 hr.
LumiGLO® is a registered trademark of Kirkegaard & Perry Laboratories.
posted June 2020
Protocol Id: 2045
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