Description
Human BAG6 (Large proline-rich protein BAG6) ELISA Kit
The Human BAG6 (Large Proline-Rich Protein BAG6) ELISA Kit is a highly sensitive and specific assay designed for the accurate measurement of BAG6 levels in human serum, plasma, and cell culture supernatants. This kit provides reliable and reproducible results, making it an excellent tool for various research applications.BAG6 is a proline-rich protein that plays a crucial role in protein quality control and degradation. It is involved in various cellular processes, including protein folding, trafficking, and degradation. Dysregulation of BAG6 has been implicated in several diseases, making it a valuable biomarker for studying conditions such as cancer, neurodegenerative disorders, and autoimmune diseases.
This ELISA kit offers researchers a powerful tool for investigating the role of BAG6 in health and disease, enabling the development of potential therapeutic strategies. Its high sensitivity and specificity make it an indispensable resource for understanding the intricate mechanisms underlying protein quality control and cellular homeostasis.
| Product Name: | Human BAG6 (Large proline-rich protein BAG6) ELISA Kit |
| Product Code: | HUFI03022 |
| Size: | 96 Assays |
| Alias: | BAG6 |
| Detection method: | Sandwich ELISA, Double Antibody |
| Application: | This immunoassay kit allows for the in vitro quantitative determination of Human BAG6 concentrations in serum plasma and other biological fluids. |
| Sensitivity: | 0.188ng/ml |
| Range: | 0.313-20ng/ml |
| Storage: | 4°C for 6 months |
| Note: | For Research Use Only |
| Recovery: | Matrices listed below were spiked with certain level of Human BAG6 and the recovery rates were calculated by comparing the measured value to the expected amount of Human BAG6 in samples. Enquire for more information. |
| Linearity: | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human BAG6 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. Enquire for more information. |
| 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 | P46379 |
| UniProt Protein Function: | BAT3: Chaperone that plays a key role in various processes such as apoptosis, insertion of tail-anchored (TA) membrane proteins to the endoplasmic reticulum membrane and regulation of chromatin. Acts in part by regulating stability of proteins and their degradation by the proteasome. Participates in endoplasmic reticulum stress-induced apoptosis via its interaction with AIFM1/AIF by regulating AIFM1/AIF stability and preventing its degradation. Also required during spermatogenesis for synaptonemal complex assembly via its interaction with HSPA2, by inhibiting polyubiquitination and subsequent proteasomal degradation of HSPA2. Required for selective ubiquitin-mediated degradation of defective nascent chain polypeptides by the proteasome. In this context, may play a role in immuno-proteasomes to generate antigenic peptides via targeted degradation, thereby playing a role in antigen presentation in immune response. Key component of the BAG6/BAT3 complex, a cytosolic multiprotein complex involved in the post-translational delivery of tail-anchored (TA) membrane proteins to the endoplasmic reticulum membrane. TA membrane proteins, also named type II transmembrane proteins, contain a single C-terminal transmembrane region. BAG6/BAT3 acts by facilitating TA membrane proteins capture by ASNA1/TRC40: it is recruited to ribosomes synthesizing membrane proteins, interacts with the transmembrane region of newly released TA proteins and transfers them to ASNA1/TRC40 for targeting to the endoplasmic reticulum membrane. Component of the BAT3 complex, at least composed of BAG6/BAT3, UBL4A and GET3/TRC35. Interacts with AIFM1, CTCFL, HSPA2 and p300/EP300. Interacts with ricin A chain. Interacts with L.pneumophila proteins Lpg2160 and LegU1. Interacts with NCR3. 3 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Chaperone; Cell cycle regulation; Apoptosis; Ubiquitin conjugating system Chromosomal Location of Human Ortholog: 6p21.3 Cellular Component: nucleoplasm; intracellular membrane-bound organelle; cytoplasm; nucleus; cytosol Molecular Function:protein binding; ubiquitin protein ligase binding; ribosome binding; Hsp70 protein binding; polyubiquitin binding Biological Process: negative regulation of proteolysis; ubiquitin-dependent protein catabolic process; synaptonemal complex assembly; internal peptidyl-lysine acetylation; protein stabilization; immune system process; chromatin modification; negative regulation of proteasomal ubiquitin-dependent protein catabolic process; regulation of cell proliferation; embryonic development; transport; spermatogenesis; brain development; cell differentiation; kidney development; DNA damage response, signal transduction by p53 class mediator resulting in induction of apoptosis; negative regulation of apoptosis; lung development |
| NCBI Summary: | This gene was first characterized as part of a cluster of genes located within the human major histocompatibility complex class III region. This gene encodes a nuclear protein that is cleaved by caspase 3 and is implicated in the control of apoptosis. In addition, the protein forms a complex with E1A binding protein p300 and is required for the acetylation of p53 in response to DNA damage. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P46379 |
| NCBI GenInfo Identifier: | 76800648 |
| NCBI Gene ID: | 7917 |
| NCBI Accession: | P46379.2 |
| UniProt Secondary Accession: | P46379,O95874, A2ADJ7, A3KQ42, A3KQ44, A6NGY6, A6PWF7 B0UX84, B4DZ12, B4E3V4, E7EMZ4, F8VXY4, |
| UniProt Related Accession: | P46379 |
| Molecular Weight: | 1132 |
| NCBI Full Name: | Large proline-rich protein BAG6 |
| NCBI Synonym Full Names: | BCL2-associated athanogene 6 |
| NCBI Official Symbol: | BAG6Â Â |
| NCBI Official Synonym Symbols: | G3; BAT3; BAG-6; D6S52EÂ Â |
| NCBI Protein Information: | large proline-rich protein BAG6; scythe; protein G3; protein Scythe; HLA-B associated transcript 3; HLA-B associated transcript-3; HLA-B-associated transcript 3; large proline-rich protein BAT3; BAG family molecular chaperone regulator 6 |
| UniProt Protein Name: | Large proline-rich protein BAG6 |
| UniProt Synonym Protein Names: | BAG family molecular chaperone regulator 6; BCL2-associated athanogene 6; BAG-6; BAG6; HLA-B-associated transcript 3; Protein G3; Protein Scythe |
| Protein Family: | BAG family molecular chaperone regulator |
| UniProt Gene Name: | BAG6Â Â |
| UniProt Entry Name: | BAG6_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. |
