Mouse Beta-1,4-galactosyltransferase 1 (B4galt1) ELISA Kit

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SKU:
MOEB2401
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P15535
Range:
0.078-5 ng/mL
ELISA Type:
Sandwich
Synonyms:
B4galt1, GGTB2, 1
Reactivity:
Mouse
Frequently bought together:

Description

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Product Name:Mouse Beta-1,4-galactosyltransferase 1 (B4galt1) ELISA Kit
Product Code:MOEB2401
Alias:Beta-1, 4-galactosyltransferase 1, Beta-1, 4-GalTase 1, Beta4Gal-T1, b4Gal-T1, UDP-Gal:beta-GlcNAc beta-1, 4-galactosyltransferase 1, UDP-galactose:beta-N-acetylglucosamine beta-1, 4-galactosyltransferase 1, B4galt1, Ggtb, Ggtb2, 2.4.1.-
Uniprot:P15535
Reactivity:Mouse
Range:0.078-5 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:B4GALT1: The Golgi complex form catalyzes the production of lactose in the lactating mammary gland and could also be responsible for the synthesis of complex-type N-linked oligosaccharides in many glycoproteins as well as the carbohydrate moieties of glycolipids. Defects in B4GALT1 are the cause of congenital disorder of glycosylation type 2D (CDG2D). CDGs are a family of severe inherited diseases caused by a defect in protein N- glycosylation. They are characterized by under-glycosylated serum proteins. These multisystem disorders present with a wide variety of clinical features, such as disorders of the nervous system development, psychomotor retardation, dysmorphic features, hypotonia, coagulation disorders, and immunodeficiency. The broad spectrum of features reflects the critical role of N-glycoproteins during embryonic development, differentiation, and maintenance of cell functions. Belongs to the glycosyltransferase 7 family. 2 isoforms of the human protein are produced by alternative initiation.
UniProt Protein Details:

Protein type:EC 2.4.1.90; Glycan Metabolism - N-glycan biosynthesis; Cell adhesion; Membrane protein, integral; Glycan Metabolism - keratan sulfate biosynthesis; EC 2.4.1.22; Motility/polarity/chemotaxis; Cell surface; EC 2.4.1.38; Transferase; Cell development/differentiation; Carbohydrate Metabolism - galactose; Glycan Metabolism - glycosphingolipid biosynthesis - lacto and neolacto series

Chromosomal Location of Human Ortholog: 9p13

Cellular Component: glycocalyx; Golgi apparatus; desmosome; extracellular space; Golgi trans cisterna; basolateral plasma membrane; brush border membrane; integral to membrane; Golgi membrane; membrane; plasma membrane; filopodium; external side of plasma membrane

Molecular Function:beta-N-acetylglucosaminylglycopeptide beta-1,4-galactosyltransferase activity; protein homodimerization activity; N-acetyllactosamine synthase activity; manganese ion binding; lactose synthase activity; cytoskeletal protein binding; beta-tubulin binding; UDP-galactosyltransferase activity; galactosyltransferase activity; alpha-tubulin binding

Biological Process: keratan sulfate metabolic process; extracellular matrix organization and biogenesis; glycosaminoglycan metabolic process; positive regulation of epithelial cell proliferation involved in wound healing; oligosaccharide biosynthetic process; positive regulation of apoptosis involved in mammary gland involution; regulation of acrosome reaction; pathogenesis; lactose biosynthetic process; negative regulation of cell proliferation; mammary gland development; development of secondary sexual characteristics; single fertilization; penetration of zona pellucida; epithelial cell development; cell adhesion; Notch signaling pathway; binding of sperm to zona pellucida; protein amino acid N-linked glycosylation; regulation of cell motility; angiogenesis involved in wound healing; post-translational protein modification; multicellular organism reproduction; cellular protein metabolic process; branching morphogenesis of a tube; keratan sulfate biosynthetic process; carbohydrate metabolic process; galactose metabolic process; protein amino acid N-linked glycosylation via asparagine; leukocyte migration; acute inflammatory response

Disease: Congenital Disorder Of Glycosylation, Type Iid

NCBI Summary:This gene is one of seven beta-1,4-galactosyltransferase (beta4GalT) genes. They encode type II membrane-bound glycoproteins that appear to have exclusive specificity for the donor substrate UDP-galactose; all transfer galactose in a beta1,4 linkage to similar acceptor sugars: GlcNAc, Glc, and Xyl. Each beta4GalT has a distinct function in the biosynthesis of different glycoconjugates and saccharide structures. As type II membrane proteins, they have an N-terminal hydrophobic signal sequence that directs the protein to the Golgi apparatus and which then remains uncleaved to function as a transmembrane anchor. By sequence similarity, the beta4GalTs form four groups: beta4GalT1 and beta4GalT2, beta4GalT3 and beta4GalT4, beta4GalT5 and beta4GalT6, and beta4GalT7. This gene is unique among the beta4GalT genes because it encodes an enzyme that participates both in glycoconjugate and lactose biosynthesis. For the first activity, the enzyme adds galactose to N-acetylglucosamine residues that are either monosaccharides or the nonreducing ends of glycoprotein carbohydrate chains. The second activity is restricted to lactating mammary tissues where the enzyme forms a heterodimer with alpha-lactalbumin to catalyze UDP-galactose + D-glucose <=> UDP + lactose. The two enzymatic forms result from alternate transcription initiation sites and post-translational processing. Two transcripts, which differ only at the 5' end, with approximate lengths of 4.1 kb and 3.9 kb encode the same protein. The longer transcript encodes the type II membrane-bound, trans-Golgi resident protein involved in glycoconjugate biosynthesis. The shorter transcript encodes a protein which is cleaved to form the soluble lactose synthase. [provided by RefSeq, Jul 2008]
UniProt Code:P15535
NCBI GenInfo Identifier:13929462
NCBI Gene ID:2683
NCBI Accession:NP_001488.2
UniProt Secondary Accession:P15535,Q12909, Q12910, Q12911, Q14456, Q14509, Q14523 B2R710, D3DRL2,
UniProt Related Accession:P15291
Molecular Weight:43920
NCBI Full Name:beta-1,4-galactosyltransferase 1
NCBI Synonym Full Names:UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1
NCBI Official Symbol:B4GALT1  
NCBI Official Synonym Symbols:GT1; GTB; CDG2D; GGTB2; B4GAL-T1; beta4Gal-T1  
NCBI Protein Information:beta-1,4-galactosyltransferase 1
UniProt Protein Name:Beta-1,4-galactosyltransferase 1
UniProt Synonym Protein Names:UDP-Gal:beta-GlcNAc beta-1,4-galactosyltransferase 1; UDP-galactose:beta-N-acetylglucosamine beta-1,4-galactosyltransferase 1
Protein Family:Beta-1,4-galactosyltransferase
UniProt Gene Name:B4GALT1  
UniProt Entry Name:B4GT1_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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