Human Complement C5 (C5) ELISA Kit
- SKU:
- HUEB0373
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- P01031
- Range:
- 0.156-10 ng/ml
- ELISA Type:
- Sandwich
- Synonyms:
- C5a, Complement Component 5a
- Reactivity:
- Human
Frequently bought together:
Description
| Product Name: | Human Complement C5 (C5) ELISA Kit |
| Product Code: | HUEB0373 |
| Alias: | Complement C5, C3 and PZP-like alpha-2-macroglobulin domain-containing protein 4, C5, CPAMD4 |
| Uniprot: | P01031 |
| Reactivity: | Human |
| Range: | 0.156-10 ng/ml |
| Detection Method: | Sandwich |
| Size: | 96 Assay |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | C5: Activation of C5 by a C5 convertase initiates the spontaneous assembly of the late complement components, C5-C9, into the membrane attack complex. C5b has a transient binding site for C6. The C5b-C6 complex is the foundation upon which the lytic complex is assembled. Defects in C5 are the cause of complement component 5 deficiency (C5D). A rare defect of the complement classical pathway associated with susceptibility to severe recurrent infections, predominantly by Neisseria gonorrhoeae or Neisseria meningitidis. An association study of C5 haplotypes and genotypes in individuals with chronic hepatitis C virus infection shows that individuals homozygous for the C5_1 haplotype have a significantly higher stage of liver fibrosis than individuals carrying at least 1 other allele (PubMed:15995705). |
| UniProt Protein Details: | Protein type:Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 9q33-q34 Cellular Component: membrane attack complex; extracellular space; extracellular region Molecular Function:protein binding; chemokine activity; endopeptidase inhibitor activity; C5a anaphylatoxin chemotactic receptor binding; receptor binding Biological Process: in utero embryonic development; positive regulation of chemotaxis; activation of MAPK activity; cytolysis; complement activation, alternative pathway; chemotaxis; glucose homeostasis; leukocyte migration during inflammatory response; complement activation; cellular calcium ion homeostasis; G-protein coupled receptor protein signaling pathway; positive regulation of angiogenesis; cell surface receptor linked signal transduction; regulation of complement activation; response to stress; innate immune response; negative regulation of dopamine secretion; inflammatory response; complement activation, classical pathway Disease: Complement Component 5 Deficiency; Eculizumab, Poor Response To |
| NCBI Summary: | The protein encoded by this gene is the fifth component of complement, which plays an important role in inflammatory and cell killing processes. This protein is comprised of alpha and beta polypeptide chains that are linked by a disulfide bridge. An activation peptide, C5a, which is an anaphylatoxin that possesses potent spasmogenic and chemotactic activity, is derived from the alpha polypeptide via cleavage with a convertase. The C5b macromolecular cleavage product can form a complex with the C6 complement component, and this complex is the basis for formation of the membrane attack complex, which includes additional complement components. Mutations in this gene cause complement component 5 deficiency, a disease where patients show a propensity for severe recurrent infections. Defects in this gene have also been linked to a susceptibility to liver fibrosis and to rheumatoid arthritis. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P01031 |
| NCBI GenInfo Identifier: | 166900096 |
| NCBI Gene ID: | 727 |
| NCBI Accession: | P01031.4 |
| UniProt Secondary Accession: | P01031,Q14CJ0, Q27I61, |
| UniProt Related Accession: | P01031 |
| Molecular Weight: | 188,305 Da |
| NCBI Full Name: | Complement C5 |
| NCBI Synonym Full Names: | complement component 5 |
| NCBI Official Symbol: | C5 |
| NCBI Official Synonym Symbols: | C5D; C5a; C5b; ECLZB; CPAMD4 |
| NCBI Protein Information: | complement C5; prepro-C5; C5a anaphylatoxin; anaphylatoxin C5a analog; C3 and PZP-like alpha-2-macroglobulin domain-containing protein 4 |
| UniProt Protein Name: | Complement C5 |
| UniProt Synonym Protein Names: | C3 and PZP-like alpha-2-macroglobulin domain-containing protein 4Cleaved into the following 4 chains:Complement C5 beta chain; Complement C5 alpha chain; C5a anaphylatoxin; Complement C5 alpha' chain |
| Protein Family: | Complement C5 |
| UniProt Gene Name: | C5 |
| UniProt Entry Name: | CO5_HUMAN |
| Component | Quantity (96 Assays) | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | -20°C |
| Lyophilized Standard | 2 | -20°C |
| Sample Diluent | 20ml | -20°C |
| Assay Diluent A | 10mL | -20°C |
| Assay Diluent B | 10mL | -20°C |
| Detection Reagent A | 120µL | -20°C |
| Detection Reagent B | 120µL | -20°C |
| Wash Buffer | 30mL | 4°C |
| Substrate | 10mL | 4°C |
| Stop Solution | 10mL | 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
*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. |
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