Description
Human Ras Related C3 Botulinum Toxin Substrate 1/RAC1 ELISA Kit
The Human Ras-Related C3 Botulinum Toxin Substrate 1 (Rac1) ELISA Kit is a powerful tool for the quantitative detection of Rac1 levels in human samples including serum, plasma, and cell culture supernatants. With exceptional sensitivity and specificity, this kit ensures reliable and accurate results for a variety of research applications. Rac1 is a key GTPase protein involved in regulating cell migration, proliferation, and cytoskeletal dynamics. Dysregulation of Rac1 activity has been implicated in various diseases, including cancer, cardiovascular diseases, and neurological disorders.
Therefore, measuring Rac1 levels can provide valuable insights into disease mechanisms and potential therapeutic targets. This ELISA kit offers a convenient and efficient solution for studying Rac1 signaling pathways and exploring its role in disease progression. Trust in the Human Rac1 ELISA Kit for precise and reproducible results, advancing your research efforts in understanding Rac1 biology and its implications in human health.
| Product Name: | Human Ras Related C3 Botulinum Toxin Substrate 1/RAC1 ELISA Kit |
| Product Code: | HUFI02979 |
| Size: | 96 Assays |
| Alias: | MGC111543, MIG5, TC-25, p21-Rac1, migRation-inducing gene 5, ras-related C3 botulinum toxin substRate 1, rho family, small GTP binding protein Rac1 |
| Detection method: | Sandwich ELISA, Double Antibody |
| Application: | This immunoassay kit allows for the in vitro quantitative determination of Human RAC1 concentrations in serum plasma and other biological fluids. |
| Sensitivity: | 0.094ng/ml |
| Range: | 0.156-10ng/ml |
| Storage: | 4°C for 6 months |
| Note: | For Research Use Only |
| Recovery: | Matrices listed below were spiked with certain level of Human RAC1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human RAC1 in samples. | ||||||||||||||||
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| Linearity: | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human RAC1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. | ||||||||||||||||
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| CV(%): | Intra-Assay: CV<8% Inter-Assay: CV<10% |
| Component | Quantity | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | 4°C for 6 months |
| Lyophilized Standard | 2 | 4°C/-20°C |
| Sample/Standard Dilution Buffer | 20ml | 4°C |
| Biotin-labeled Antibody(Concentrated) | 120ul | 4°C (Protect from light) |
| Antibody Dilution Buffer | 10ml | 4°C |
| HRP-Streptavidin Conjugate(SABC) | 120ul | 4°C (Protect from light) |
| SABC Dilution Buffer | 10ml | 4°C |
| TMB Substrate | 10ml | 4°C (Protect from light) |
| Stop Solution | 10ml | 4°C |
| Wash Buffer(25X) | 30ml | 4°C |
| Plate Sealer | 5 | - |
Other materials and equipment required:
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Incubator
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
| Uniprot | P63000 |
| UniProt Protein Function: | RAC1: a plasma membrane-associated member of the Rho-GTPase family. Plays a key role in cytoskeletal reorganization, membrane trafficking, transcriptional regulation and cell growth and development. GTP binding stimulates its activity. Phosphorylation by Akt may inhibit GTP binding of Rac1, therefore attenuating the downstream signal transduction pathway. Found in a trimeric complex composed of DOCK1 and ELMO1, which plays a central role in phagocytosis of apoptotic cells. Two alternatively spliced isoforms have been described. |
| UniProt Protein Details: | Protein type:Motility/polarity/chemotaxis; G protein; G protein, monomeric, Rho; G protein, monomeric Chromosomal Location of Human Ortholog: 7p22 Cellular Component: extrinsic to plasma membrane; Golgi membrane; focal adhesion; membrane; lamellipodium; cytoplasm; plasma membrane; melanosome; actin filament; trans-Golgi network; cytosol; phagocytic cup Molecular Function:GTPase activity; protein binding; enzyme binding; GTP binding; GTP-dependent protein binding; Rho GDP-dissociation inhibitor binding; thioesterase binding; protein kinase binding; Rab GTPase binding Biological Process: viral reproduction; nerve growth factor receptor signaling pathway; metabolic process; positive regulation of apoptosis; cell motility involved in cell locomotion; regulation of cell migration; small GTPase mediated signal transduction; positive regulation of stress fiber formation; cell adhesion; bone resorption; platelet activation; anatomical structure morphogenesis; mast cell chemotaxis; dendrite morphogenesis; positive regulation of Rho protein signal transduction; regulation of defense response to virus by virus; negative regulation of interleukin-23 production; T cell costimulation; cerebral cortex radially oriented cell migration; actin cytoskeleton organization and biogenesis; axon guidance; cell-matrix adhesion; localization within membrane; positive regulation of focal adhesion formation; actin filament polymerization; response to wounding; ephrin receptor signaling pathway; inflammatory response; regulation of hydrogen peroxide metabolic process; lamellipodium biogenesis; embryonic olfactory bulb interneuron precursor migration; intercellular junction assembly and maintenance; Wnt receptor signaling pathway, planar cell polarity pathway; positive regulation of phosphoinositide 3-kinase activity; engulfment of apoptotic cell; cell proliferation; G-protein coupled receptor protein signaling pathway; hyperosmotic response; positive regulation of actin filament polymerization; organization of an anatomical structure; auditory receptor cell morphogenesis; ruffle organization and biogenesis; innate immune response; negative regulation of receptor-mediated endocytosis; positive regulation of protein amino acid phosphorylation; vascular endothelial growth factor receptor signaling pathway; blood coagulation; cell motility; positive regulation of DNA replication |
| NCBI Summary: | The protein encoded by this gene is a GTPase which belongs to the RAS superfamily of small GTP-binding proteins. Members of this superfamily appear to regulate a diverse array of cellular events, including the control of cell growth, cytoskeletal reorganization, and the activation of protein kinases. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Mar 2009] |
| UniProt Code: | P63000 |
| NCBI GenInfo Identifier: | 51702787 |
| NCBI Gene ID: | 5879 |
| NCBI Accession: | P63000.1 |
| UniProt Secondary Accession: | P63000,O95501, P15154, Q3Y4D3, Q5JAA8, Q9BTB4, |
| UniProt Related Accession: | P63000,AAB22206 |
| Molecular Weight: | 192 |
| NCBI Full Name: | Ras-related C3 botulinum toxin substrate 1 |
| NCBI Synonym Full Names: | ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1) |
| NCBI Official Symbol: | RAC1Â Â |
| NCBI Official Synonym Symbols: | MIG5; Rac-1; TC-25; p21-Rac1Â Â |
| NCBI Protein Information: | ras-related C3 botulinum toxin substrate 1; ras-like protein TC25; cell migration-inducing gene 5 protein |
| UniProt Protein Name: | Ras-related C3 botulinum toxin substrate 1 |
| UniProt Synonym Protein Names: | Cell migration-inducing gene 5 protein; Ras-like protein TC25; p21-Rac1 |
| Protein Family: | Ras-related protein |
| UniProt Gene Name: | RAC1Â Â |
| UniProt Entry Name: | RAC1_HUMAN |
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.
| Step | Protocol |
| 1. | Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells! |
| 2. | Aliquot 0.1ml standard solutions into the standard wells. |
| 3. | Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well. |
| 4. | Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells. |
| 5. | Seal the plate with a cover and incubate at 37 °C for 90 min. |
| 6. | Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2. |
| 7. | Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall. |
| 8. | Seal the plate with a cover and incubate at 37°C for 60 min. |
| 9. | Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash. |
| 10. | Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min. |
| 11. | Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min. |
| 12. | Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color. |
| 13. | Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately. |
| 14. | Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution. |
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. |
