Whole blood collected from 7 donors was washed in RPMI 1640 four times to remove plasma, platelets, and leukocytes. The number of erythrocytes was determined by using a cell counting chamber. Erythrocytes were incubated for 2 h at 37°C with or without 2.5 μM EHop-016 and then washed. In each sample, the Rac1 activation state was measured with the G-LISA Rac1 activation assay luminescence-based biochemical kit (Cytoskeleton). Luminescence intensity was assessed with a BioTek Synergy HT plate reader. These experiments were performed in three biological replicates.
Rac1 g lisa activation assay
Manufactured by Cytoskeleton
Sourced in United States
The Rac1 G-LISA Activation Assay is a laboratory equipment product designed to detect and quantify the activation of the Rac1 protein, a member of the Rho family of small GTPases. The assay utilizes a Rac1-specific binding protein immobilized on a 96-well plate to capture and measure the levels of activated Rac1 in cell or tissue lysates.
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Lab products found in correlation
6 protocols using rac1 g lisa activation assay
1
Rac1 Activation in Erythrocytes
2
Rac1 Activity Assay in Prostate Cancer Cells
3
Quantifying Rac1 Activation in Rat NAc
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At specified time points, the rats were sacrificed by decapitation, and the brain was quickly removed and snap frozen in 2-Methylbutane (Sigma Aldrich, St. Louis, MO) for 1 min. Using a brain matrix, 2-mm thick brain slices were prepared and tissue punches containing the NAc were extracted. The punched tissue was homogenized in lysis buffer (4ºC) and then centrifuged at 14000 g for 2 min at 4ºC. The supernatant was collected and snap frozen. Lysate was stored at −80ºC. Rac1-GTP was quantified with the Rac1 G-LISA Activation Assay (Absorbance Based) (Cytoskeleton, Denver) according to the manufacture instructions. Rac1-GTP levels were then assessed by the absorbance of each sample at 490 nm.
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Rac1 Activity Assay in BMDMs
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BMDMs were differentiated, as described previously, and were treated with apoptotic cells for 30 min. BMDMs were washed 3 times in ice-cold PBS and harvested, according to the Rac1 GLISA activation assay manufacturer protocol (Cytoskeleton, Denver, CO, USA). Briefly, BMDMs were treated with lysis buffer, scraped, and snap-frozen in liquid nitrogen until used. Absorbance was read at 490 nm using a Tecan plate reader. The data were reported as relative absorbance.
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Quantifying Rac1 Activation in Rat NAc
6
Quantifying Rac1 Activation in ER-Targeted Cells
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For measuring the level of activation of Rac1, luminescence‐based Rac1 G‐LISA Activation Assay (Cytoskeleton Inc. #BK126) kit was used. The assay was carried out following the protocol (freely available at manufacturer's website) provided with the kit without any modifications. Cell lysates prepared from HeLa cells stably overexpressing ER‐targeted Rac1‐GAP, KDEL R2, or ER‐targeted catalytically inactive Rac1‐GAP were used for the measurements.
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