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Question 1: At which step can the assay be stopped?
Answer 1: The assay cannot be stopped until after the 100, 000 x g spin for 1 hour at 37°C. After this high speed centrifugation, the supernatant (G-actin) can be mixed with SDS loading buffer and frozen for later use. The pellet (F-actin) should be resuspended with a depolymerizing agent and water and then mixed with SDS loading buffer and frozen for later use. Upon freezing, F-actin depolymerizes, so it is necessary to separate the F-actin from the G-actin before freezing samples to isolate samples for an accurate measurement of F-actin and G-actin ratios.
Question 2: How sensitive is this assay?
Answer 2: The assay can detect as small as a 15% shift in G-actin to F-actin ratio. Each condition should be performed in duplicate and repeated several times as assay reproducibility can vary by 10-20% between experiments.
Introduction
Panel 1: In untreated Swiss 3T3 cells, 45% of actin is soluble G-actin (1S) and 55% is insoluble F-actin (1P). This agrees with published data (3).Panel 2: In Swiss 3T3 cells treated with the actin polymerizing drug jasplakinolide, only 5% of actin remains in the soluble G-actin fraction (2S) while 95% is found in the insoluble F-actin pellet fraction (2P). Lanes 50, 20 and 10 represents 50ng, 20ng and 10 ng of G-actin standard. M represents molecular weight markers (molecular weights are shown to the right of the blot).
Note: The presented information and documents (Manual, Product Datasheet, Safety Datasheet and Certificate of Analysis) correspond to our latest update and should serve for orientational purpose only. We do not guarantee the topicality. We would kindly ask you to make a request for specific requirements, if necessary.
All products are intended for research use only (RUO). Not for human, veterinary or therapeutic use.
BK037.pdf
BK037-datasheet.pdf
BK037-safety-datasheet.pdf
Delivery expected until 8/7/2025
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