Description
Human Cytosolic beta-glucosidase (GBA3) ELISA Kit
The Human Cytosolic Beta-Glucosidase (GBA3) ELISA Kit is specifically designed for the precise detection of GBA3 levels in human serum, plasma, and cell culture supernatants. This kit offers superior sensitivity and specificity, ensuring accurate and consistent results for a variety of research applications.GBA3 is an important enzyme involved in various cellular functions, including carbohydrate metabolism and glycosylation processes. Dysregulation of GBA3 has been linked to metabolic disorders, lysosomal storage diseases, and other pathological conditions, highlighting its potential as a diagnostic and therapeutic target.
With its advanced technology and user-friendly protocols, the Human Cytosolic Beta-Glucosidase (GBA3) ELISA Kit is a valuable tool for studying GBA3 biology and its implications in human health and disease. Get reliable data and valuable insights with this innovative assay kit from Assay Genie.
| Product Name: | Human Cytosolic beta-glucosidase (GBA3) ELISA Kit |
| SKU: | HUEB1798 |
| Size: | 96T |
| Target: | Human Cytosolic beta-glucosidase (GBA3) |
| Synonyms: | Cytosolic beta-glucosidase-like protein 1, Cytosolic glycosylceramidase, Glucosidase beta acid 3, Glucosylceramidase beta 3, Klotho-related protein, Cytosolic GCase, KLrP, CBG, CBGL1 |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Human |
| Detection Range: | 0.156-10ng/mL |
| Sensitivity: | 0.1ng/mL |
| Intra CV: | 5.2% | ||||||||||||||||||||
| Inter CV: | 8.3% | ||||||||||||||||||||
| Linearity: |
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| Recovery: |
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| Function: | Neutral cytosolic beta-glycosidase with a broad substrate specificity that could play a role in the catabolism of glycosylceramides (PubMed:11389701, PubMed:11784319, PubMed:20728381, PubMed:26724485, PubMed:17595169). Has a significant glucosylceramidase activity in vitro (PubMed:26724485, PubMed:17595169). However, that activity is relatively low and its significance in vivo is not clear (PubMed:26724485, PubMed:17595169, PubMed:20728381). Also able to hydrolyze galactosylceramide/GalCer, glucosylsphingosine/GlcSph and galactosylsphingosine/GalSph (PubMed:17595169). However, the in vivo relevance of these activities is unclear (PubMed:17595169). It can also hydrolyze a broad variety of dietary glycosides including phytoestrogens, flavonols, flavones, flavanones and cyanogens in vitro and could therefore play a role in the metabolism of xenobiotics (PubMed:11784319). Could also play a role in the catabolism of cytosolic sialyl free N-glycans (PubMed:26193330). |
| Uniprot: | Q9H227 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant human Cytosolic beta-glucosidase |
| Sub Unit: | May interact with NEU2. |
| Subcellular Location: | Cytoplasm Cytosol |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | GBA3: Glycosidase probably involved in the intestinal absorption and metabolism of dietary flavonoid glycosides. Able to hydrolyze a broad variety of glycosides including phytoestrogens, flavonols, flavones, flavanones and cyanogens. Possesses beta- glycosylceramidase activity and may be involved in a nonlysosomal catabolic pathway of glycosylceramide. Belongs to the glycosyl hydrolase 1 family. Klotho subfamily. 2 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Carbohydrate Metabolism - starch and sucrose; Hydrolase; EC 3.2.1.21; Other Amino Acids Metabolism - cyanoamino acid Chromosomal Location of Human Ortholog: 4p15.2 Cellular Component: cytosol Molecular Function:beta-galactosidase activity; beta-glucosidase activity; glucosylceramidase activity; glycosylceramidase activity Biological Process: glycoside catabolic process; glycosphingolipid metabolic process; glycosylceramide catabolic process |
| NCBI Summary: | The protein encoded by this gene is an enzyme that can hydrolyze several types of glycosides. This gene is a polymorphic pseudogene, with the most common allele being the functional allele that encodes the full-length protein. Some individuals, as represented by the reference genome allele, contain a single nucleotide polymorphism that results in a premature stop codon in the coding region, and therefore this allele is pseudogenic due to the failure to produce a functional full-length protein. Alternative splicing of this gene results in multiple transcript variants. [provided by RefSeq, Mar 2013] |
| UniProt Code: | Q9H227 |
| NCBI GenInfo Identifier: | 77416427 |
| NCBI Gene ID: | 57733 |
| NCBI Accession: | Q9H227.2 |
| UniProt Secondary Accession: | Q9H227,Q32LY7, Q3MIH4, Q53GG8, Q6NSF4, Q8NHT8, Q9H3T4 Q9H4C6, |
| UniProt Related Accession: | Q9H227 |
| Molecular Weight: | 18,265 Da |
| NCBI Full Name: | Cytosolic beta-glucosidase |
| NCBI Synonym Full Names: | glucosylceramidase beta 3 (gene/pseudogene) |
| NCBI Official Symbol: | GBA3 |
| NCBI Official Synonym Symbols: | CBG; GLUC; KLRP; CBGL1 |
| NCBI Protein Information: | cytosolic beta-glucosidase |
| UniProt Protein Name: | Cytosolic beta-glucosidase |
| UniProt Synonym Protein Names: | Cytosolic beta-glucosidase-like protein 1 |
| Protein Family: | Cytosolic beta-glucosidase |
| UniProt Gene Name: | GBA3 |
| UniProt Entry Name: | GBA3_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. |
