Porcine Integrin beta-1 (ITGB1) ELISA Kit

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SKU:
PREB0566
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q9GLP0
ELISA Type:
Sandwich
Reactivity:
Pig
Frequently bought together:

Description

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Product Name:Porcine Integrin beta-1 (ITGB1) ELISA Kit
Product Code:PREB0566
Alias:Integrin beta-1, Fibronectin receptor subunit beta, VLA-4 subunit beta, ITGB1, CD29
Uniprot:Q9GLP0
Reactivity:Pig
Range:Please contact us for more information
Detection Method:Sandwich
Size:96 Assay
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:ITGB1: an integral membrane protein that heterodimerizes with an alpha-3 chain, forming a receptor for many extracellular-matrix proteins including fibronectin, laminin, collagen, epiligrin and thrombospondin. . Beta 1 integrins recognize the amino-acid motif RGD in a wide array of ligands. Five alternatively spliced variants with alternate carboxy termini have been described. Two alternatively spliced isoforms have been described. Isoform beta-1a is widely expressed; other isoforms are generally expressed with a more restricted distribution. Isoform beta-1b is expressed in skin, liver, skeletal muscle, cardiac muscle, placenta, umbilical vein endothelial cells, neuroblastoma cells, lymphoma cells, hepatoma cells and astrocytoma cells. Isoforms beta-1c and beta-1c-2 are expressed in muscle, kidney, liver, placenta, cervical epithelium, umbilical vein endothelial cells, fibroblast cells, embryonal kidney cells, platelets and several blood cell lines. Isoform beta-c-2, rather than isoform beta-1c, is selectively expressed in primary t-cells. Isoform beta-1c is expressed in nonproliferating and differentiated prostate gland epithelial cells. Isoform beta-1d is expressed specifically in striated muscle (skeletal and cardiac muscle).Protein type: Motility/polarity/chemotaxis; Cell adhesion; Receptor, misc.; Membrane protein, integral; Cell surfaceChromosomal Location of Human Ortholog: 10p11.2Cellular Component: focal adhesion; cell surface; dendritic spine; acrosome; lipid raft; ruffle; recycling endosome; hemidesmosome; membrane; lamellipodium; cytoplasm; plasma membrane; melanosome; basement membrane; integrin complex; neuromuscular junction; sarcolemma; receptor complex; external side of plasma membrane; cleavage furrow; filopodiumMolecular Function: protein domain specific binding; protease binding; metal ion binding; laminin binding; actin binding; alpha-actinin binding; protein kinase binding; peptide binding; viral receptor activity; integrin binding; protein binding; protein heterodimerization activity; fibronectin binding; cell adhesion molecule binding; glycoprotein bindingBiological Process: extracellular matrix organization and biogenesis; maternal process involved in pregnancy; regulation of cell cycle; axon extension; positive regulation of apoptosis; mesodermal cell differentiation; positive regulation of endocytosis; cardiac muscle cell differentiation; leukocyte adhesion; transforming growth factor beta receptor signaling pathway; germ cell migration; tissue homeostasis; response to drug; dendrite morphogenesis; protein transport within lipid bilayer; positive regulation of peptidyl-tyrosine phosphorylation; negative regulation of neuron differentiation; cell migration during sprouting angiogenesis; B cell differentiation; cell-cell adhesion mediated by integrin; response to activity; leukocyte migration; regulation of G-protein coupled receptor protein signaling pathway; G1/S transition of mitotic cell cycle; axon guidance; entry of virus into host cell; cell-matrix adhesion; cell fate specification; negative regulation of cell proliferation; positive regulation of MAPKKK cascade; positive regulation of cell proliferation; visual learning; negative regulation of cell projection organization and biogenesis; integrin-mediated signaling pathway; regulation of immune response; cell migration; in utero embryonic development; sarcomere organization; cell-substrate adhesion; formation of radial glial scaffolds; cellular calcium ion homeostasis; heterotypic cell-cell adhesion; negative regulation of Rho protein signal transduction; calcium-independent cell-matrix adhesion; stress fiber formation; cellular defense response; homophilic cell adhesion; blood coagulation; leukocyte tethering or rolling; positive regulation of cell migration
UniProt Protein Details:
NCBI Summary:Integrins are heterodimeric proteins made up of alpha and beta subunits. At least 18 alpha and 8 beta subunits have been described in mammals. Integrin family members are membrane receptors involved in cell adhesion and recognition in a variety of processes including embryogenesis, hemostasis, tissue repair, immune response and metastatic diffusion of tumor cells. This gene encodes a beta subunit. Multiple alternatively spliced transcript variants which encode different protein isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:Q9GLP0
NCBI GenInfo Identifier:19743813
NCBI Gene ID:3688
NCBI Accession:NP_002202.2
UniProt Secondary Accession:Q9GLP0,Q9GLP0
UniProt Related Accession:Q9GLP0,P05556
Molecular Weight:88,415 Da
NCBI Full Name:integrin beta-1 isoform 1A
NCBI Synonym Full Names:integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12)
NCBI Official Symbol:ITGB1  
NCBI Official Synonym Symbols:CD29; FNRB; MDF2; VLAB; GPIIA; MSK12; VLA-BETA  
NCBI Protein Information:integrin beta-1; glycoprotein IIa; integrin VLA-4 beta subunit; very late activation protein, beta polypeptide
UniProt Protein Name:Integrin beta-1
UniProt Synonym Protein Names:Fibronectin receptor subunit beta; Glycoprotein IIa; GPIIA; VLA-4 subunit beta
Protein Family:Integrin
UniProt Gene Name:ITGB1  
UniProt Entry Name:ITB1_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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