The intracellular amounts of total RhoA and RhoA‐GTP were determined by using the total RhoA ELISA and G protein‐linked (G‐LISA) assays (Cytoskeleton, Inc., Denver, CO, USA) according to the manufacturer's instructions. Briefly, cells were washed with cold PBS and homogenized gently in ice‐cold lysis buffer. 20 μl was removed for protein quantification in order to adjust sample concentration to 0.5 mg/ml. After adding an equal volume of binding buffer, triplicate assays were performed using 1.5 μg protein per well. Samples were incubated for 30 min and then washed three times with washing buffer. Antigen‐presenting buffer was added for 2 min before removal; samples were then incubated with 1:250 dilution of anti‐RhoA antibody at room temperature for 45 min, washed three times, and incubated with secondary antibodies for another 45 min. HRP detection reagent was added and signal was read by measuring absorbance at 490 nm using a microplate spectrometer.
Total rhoa elisa
Manufactured by Cytoskeleton
Sourced in United States
The Total RhoA ELISA is a quantitative immunoassay designed to measure the total levels of the RhoA protein in cell and tissue lysates. The assay utilizes a sandwich ELISA format with specific antibodies to capture and detect RhoA. The resulting signal is proportional to the amount of RhoA present in the sample.
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Lab products found in correlation
Rhoa g lisa kit, by Cytoskeleton (2 mentions)
G lisa rhoa activation kit, by Cytoskeleton (1 mentions)
Active rho pull down detection kit, by Thermo Fisher Scientific (1 mentions)
Rabbit anti rhoa antibody, by Cell Signaling Technology (1 mentions)
Anti rabbit horseradish peroxidase antibody, by Cell Signaling Technology (1 mentions)
Rhoa activation assay biochem kit, by Cytoskeleton (1 mentions)
Canto 2, by BD (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
4 protocols using total rhoa elisa
1
Quantifying Intracellular RhoA and RhoA-GTP
2
Quantifying Rho Activation Dynamics
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Rho activation was measured by Western blot analysis following pull-down of active Rho with an Active-Rho pull-down/detection kit as per the manufacturer's instructions (Thermo-Fisher Scientific, Waltham, MA) and compared to total Rho. Rho activity was also measured using a G-LISA RhoA Activation Kit and a Total RhoA ELISA as per the manufacturer's instructions (Cytoskeleton Inc.).
3
Quantifying RhoA Activity in 2D and 3D
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HT1080 cells were plated on 2D substrates and inside 3D collagen I matrices and allowed to incubate for 48 h, which was the total duration of the motility experiments. A RhoA G-LISA kit (Cytoskeleton, Denver, CO, USA) was used to assess RhoA activity according to the manufacturer’s instructions. The amount of total RhoA was assessed using 12% SDS-PAGE and a more sensitive total RhoA ELISA (Cytoskeleton). Total RhoA blots used the same lysates used in the G-LISA assay. SDS-PAGE blots were probed with rabbit anti-RhoA antibody (Cell Signaling Technology) followed by anti-rabbit horseradish peroxidase antibody (Cell Signaling Technology).
4
FACS Analysis and RhoA Activation
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For FACS analysis, non-fixed cells were detached using 0.05% Trypsin/EDTA (Invitrogen), washed 1x in PBS, washed 2x in Tris-Buffer/1%BSA at 4 C and stained with the indicated primary antibodies followed by fluorescently-conjugated secondary antibodies for 30 min each in Tris-Buffer/5% BSA on ice. Cells were immediately analyzed by FACS (BD Canto II). FACS staining was quantitated using the FlowJo (Tree Star Inc) software. The fluorescence intensity is displayed on the X-axis (divided into 256 bins). The % of Max on the Y-axis represents the number of cells in each bin on the X-axis (FlowJo uses an arbitrary number of 256 bins) divided by the number of cells in the bin that contains the largest number of cells.
RhoA Activation Assays 3T3 cells were serum-starved overnight and treated for 20 min with 1 mM Nogo-A-D20 or Nogo-A-D21 control protein. Pulldown of activated RhoA-GTP was subsequently performed using the RhoA Activation Assay Biochem Kit according to the manufacturer's instructions (Cytoskeleton, Inc.). Alternatively, RhoA activation was assessed using the total RhoA ELISA and RhoA G-LISA kit according to the manufacturer's instructions (Cytoskeleton, Inc.). Levels of activated RhoA were normalized to total RhoA levels for each biological replicate.
RhoA Activation Assays 3T3 cells were serum-starved overnight and treated for 20 min with 1 mM Nogo-A-D20 or Nogo-A-D21 control protein. Pulldown of activated RhoA-GTP was subsequently performed using the RhoA Activation Assay Biochem Kit according to the manufacturer's instructions (Cytoskeleton, Inc.). Alternatively, RhoA activation was assessed using the total RhoA ELISA and RhoA G-LISA kit according to the manufacturer's instructions (Cytoskeleton, Inc.). Levels of activated RhoA were normalized to total RhoA levels for each biological replicate.
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