The Rat Transforming Protein RHOA (RHOA) ELISA Kit is specifically designed for the accurate quantification of RHOA levels in rat samples including serum, plasma, and cell culture supernatants. With its high sensitivity and specificity, this kit ensures precise and reliable results, making it a valuable tool for various research applications.RHOA is a critical signaling protein involved in cell migration, adhesion, and cytoskeletal organization.
Dysregulation of RHOA has been linked to various diseases including cancer, cardiovascular disorders, and neurological conditions. Therefore, measuring RHOA levels can provide valuable insights into disease mechanisms and potential therapeutic targets.Overall, the Rat Transforming Protein RHOA (RHOA) ELISA Kit is an essential tool for researchers studying the role of RHOA in disease biology and developing innovative treatment strategies.
Product Name:
Rat Transforming protein RhoA (Rhoa) ELISA Kit
SKU:
RTEB1669
Size:
96T
Target:
Rat Transforming protein RhoA (Rhoa)
Synonyms:
Arha, Arha2
Assay Type:
Sandwich
Detection Method:
ELISA
Reactivity:
Rat
Detection Range:
0.156-10ng/mL
Sensitivity:
0.043ng/mL
Intra CV:
4.3%
Inter CV:
7.5%
Linearity:
Sample
1:2
1:4
1:8
1:16
Serum(N=5)
98-108%
96-106%
100-112%
92-102%
EDTA Plasma(N=5)
107-118%
97-97%
97-107%
100-110%
Heparin Plasma(N=5)
89-99%
92-104%
81-91%
103-115%
Recovery:
Sample Type
Average(%)
Recovery Range(%)
Serum
96
90-102
Plasma
98
92-104
Function:
Regulates a signal transduction pathway linking plasma membrane receptors to the assembly of focal adhesions and actin stress fibers. Involved in a microtubule-dependent signal that is required for the myosin contractile ring formation during cell cycle cytokinesis. Plays an essential role in cleavage furrow formation. Required for the apical junction formation of keratinocyte cell-cell adhesion. May be an activator of PLCE1. Activated by ARHGEF2, which promotes the exchange of GDP for GTP. Essential for the SPATA13-mediated regulation of cell migration and adhesion assembly and disassembly. The MEMO1-RHOA-DIAPH1 signaling pathway plays an important role in ERBB2-dependent stabilization of microtubules at the cell cortex. It controls the localization of APC and CLASP2 to the cell membrane, via the regulation of GSK3B activity. In turn, membrane-bound APC allows the localization of the MACF1 to the cell membrane, which is required for microtubule capture and stabilization (By similarity). Regulates KCNA2 potassium channel activity by reducing its location at the cell surface in response to CHRM1 activation; promotes KCNA2 endocytosis (PubMed:9635436).
Uniprot:
P61589
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant rat Transforming protein RhoA
Sub Unit:
Binds PRKCL1, ROCK1 and ROCK2. Interacts with ARHGEF2, ARHGEF3, NET1 and RTKN. Interacts with PLCE1 and AKAP13. Interacts with ARHGEF28. Interacts with DIAPH1. Interacts (in the constitutively activated, GTP-bound form) with DGKQ. Interacts with RACK1; enhances RHOA activation. Interacts with PKP4; the interaction is detected at the midbody. Interacts (GTP-bound form preferentially) with PKN2; the interaction stimulates autophosphorylation and phosphorylation of PKN2 (By similarity). Interacts with ARHGDIA; this interaction inactivates and stabilizes RHOA. Interacts with ARHGDIB (By similarity). Interacts (GTP-bound form) with KCNA2 (via cytoplasmic N-terminal domain) (PubMed:9635436).
Research Area:
Signal Transduction
Subcellular Location:
Cell membrane Lipid-anchor Cytoplasmic side Cytoplasm Cytoskeleton Cleavage furrow Cytoplasm Cell cortex Midbody Cell projection Lamellipodium Translocates to the equatorial region before furrow formation in a ECT2-dependent manner. Localizes to the equatorial cell cortex (at the site of the presumptive furrow) in early anaphase in a activated form and in a myosin- and actin-independent manner. Localized to cell-cell contacts in calcium-treated keratinocytes (By similarity).
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
RHOA: a small G protein of the Rho family. Regulates a signal transduction pathway linking plasma membrane receptors to the assembly of focal adhesions and actin stress fibers. Controls the reorganization of actins into podosomes. Serves as a target for the yopT cysteine peptidase from Yersinia pestis, vector of the plague, and Yersinia pseudotuberculosis, which causes gastrointestinal disorders.Protein type: G protein, monomeric, Rho; Motility/polarity/chemotaxis; G protein, monomeric; G protein; OncoproteinCellular Component: apical junction complex; focal adhesion; mitochondrion; cell cortex; cytosol; cytoskeleton; membrane; lamellipodium; axon; cytoplasm; plasma membrane; midbody; nucleus; cleavage furrowMolecular Function: GTPase activity; protein domain specific binding; protein binding; GDP binding; myosin binding; GTP binding; Rho GDP-dissociation inhibitor bindingBiological Process: radial glial cell differentiation in the forebrain; positive regulation of cell adhesion; positive regulation of translation; metabolic process; cell-matrix adhesion; response to glucocorticoid stimulus; regulation of dendrite development; cell morphogenesis; positive regulation of caspase activity; negative chemotaxis; regulation of cell migration; regulation of osteoblast proliferation; substantia nigra development; small GTPase mediated signal transduction; positive regulation of neuron apoptosis; positive regulation of stress fiber formation; stress-activated protein kinase signaling pathway; response to glucose stimulus; negative regulation of neuron apoptosis; positive regulation of smooth muscle contraction; cell differentiation; cell adhesion; response to drug; positive regulation of cytokinesis; negative regulation of steroid hormone receptor signaling pathway; skeletal muscle development; positive regulation of I-kappaB kinase/NF-kappaB cascade; apical junction assembly; regulation of actin polymerization and/or depolymerization; cytoskeleton organization and biogenesis; negative regulation of I-kappaB kinase/NF-kappaB cascade; regulation of calcium ion transport; response to amino acid stimulus; positive regulation of cell growth; regulation of transcription from RNA polymerase II promoter; negative regulation of neuron differentiation; cerebral cortex cell migration; response to ethanol; positive regulation of actin filament polymerization; response to mechanical stimulus; androgen receptor signaling pathway; response to hypoxia; stress fiber formation; positive regulation of vasoconstriction; positive regulation of neuron differentiation; actin cytoskeleton organization and biogenesis; neurite morphogenesis; positive regulation of cell migration
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
Step
1.
Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.
Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.
Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.
Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.
Repeat the wash process for five times as conducted in step 3.
6.
Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.
Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.
Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.
After experiment, store all reagents according to the specified storage temperature respectively until their expiry.
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
Sample Type
Protocol
Serum
If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma
Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid
Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant
Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates
Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates
The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates
Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk
Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.
