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Human Platelet glycoprotein Ib alpha chain (GP1BA) ELISA Kit

SKU:
HUEB1892
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P07359
Range:
15.6-1000 pg/mL
ELISA Type:
Sandwich
Reactivity:
Human
€649
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Description

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Product Name:Human Platelet glycoprotein Ib alpha chain (GP1BA) ELISA Kit
Product Code:HUEB1892
Alias:Platelet glycoprotein Ib alpha chain, GP-Ib alpha, GPIb-alpha, GPIbA, Glycoprotein Ibalpha, Antigen CD42b-alpha, GP1BA, CD42b
Uniprot:P07359
Reactivity:Human
Range:15.6-1000 pg/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:GPIbA: GP-Ib, a surface membrane protein of platelets, participates in the formation of platelet plugs by binding to the A1 domain of vWF, which is already bound to the subendothelium. Genetic variations in GP1BA may be a cause of susceptibility to non-arteritic anterior ischemic optic neuropathy (NAION). NAION is an ocular disease due to ischemic injury to the optic nerve. It usually affects the optic disk and leads to visual loss and optic disk swelling of a pallid nature. Visual loss is usually sudden, or over a few days at most and is usually permanent, with some recovery possibly occurring within the first weeks or months. Patients with small disks having smaller or non-existent cups have an anatomical predisposition for non-arteritic anterior ischemic optic neuropathy. As an ischemic episode evolves, the swelling compromises circulation, with a spiral of ischemia resulting in further neuronal damage. Defects in GP1BA are a cause of Bernard-Soulier syndrome (BSS); also known as giant platelet disease (GPD). BSS patients have unusually large platelets and have a clinical bleeding tendency. Defects in GP1BA are the cause of benign mediterranean macrothrombocytopenia (BMM); also known as autosomal dominant benign Bernard-Soulier syndrome. BMM is characterized by mild or no clinical symptoms, normal platelet function, and normal megakaryocyte count. Defects in GP1BA are the cause of pseudo-von Willebrand disease (VWDP). A bleeding disorder is caused by an increased affinity of GP-Ib for soluble vWF resulting in impaired hemostatic function due to the removal of vWF from the circulation.
UniProt Protein Details:

Protein type:Membrane protein, integral; Cell surface; Cell adhesion

Chromosomal Location of Human Ortholog: 17p13.2

Cellular Component: anchored to external side of plasma membrane; cell surface; membrane; integral to plasma membrane; plasma membrane

Molecular Function:protein binding; thrombin receptor activity

Biological Process: platelet activation; fibrinolysis; cell surface receptor linked signal transduction; regulation of blood coagulation; cell morphogenesis; cell adhesion; blood coagulation; blood coagulation, intrinsic pathway

Disease: Pseudo-von Willebrand Disease; Bernard-soulier Syndrome; Bernard-soulier Syndrome, Type A2, Autosomal Dominant; Nonarteritic Anterior Ischemic Optic Neuropathy, Susceptibility To

NCBI Summary:Glycoprotein Ib (GP Ib) is a platelet surface membrane glycoprotein composed of a heterodimer, an alpha chain and a beta chain, that is linked by disulfide bonds. The Gp Ib functions as a receptor for von Willebrand factor (VWF). The complete receptor complex includes noncovalent association of the alpha and beta subunits with platelet glycoprotein IX and platelet glycoprotein V. The binding of the GP Ib-IX-V complex to VWF facilitates initial platelet adhesion to vascular subendothelium after vascular injury, and also initiates signaling events within the platelet that lead to enhanced platelet activation, thrombosis, and hemostasis. This gene encodes the alpha subunit. Mutations in this gene result in Bernard-Soulier syndromes and platelet-type von Willebrand disease. The coding region of this gene is known to contain a polymophic variable number tandem repeat (VNTR) domain that is associated with susceptibility to nonarteritic anterior ischemic optic neuropathy. [provided by RefSeq, Oct 2013]
UniProt Code:P07359
NCBI GenInfo Identifier:121531
NCBI Gene ID:2811
NCBI Accession:P07359.1
UniProt Secondary Accession:P07359,Q14441, Q16469, Q8N1F3, Q8NG39, Q9HDC7, Q9UEK1 Q9UQS4,
UniProt Related Accession:P07359
Molecular Weight:17.4kDa
NCBI Full Name:Platelet glycoprotein Ib alpha chain
NCBI Synonym Full Names:glycoprotein Ib (platelet), alpha polypeptide
NCBI Official Symbol:GP1BA  
NCBI Official Synonym Symbols:BSS; GP1B; VWDP; CD42B; GPIbA; BDPLT1; BDPLT3; DBPLT3; CD42b-alpha  
NCBI Protein Information:platelet glycoprotein Ib alpha chain; GP-Ib alpha; antigen CD42b-alpha; platelet membrane glycoprotein 1b-alpha subunit
UniProt Protein Name:Platelet glycoprotein Ib alpha chain
UniProt Synonym Protein Names:Antigen CD42b-alpha
Protein Family:Envelope glycoprotein
UniProt Gene Name:GP1BA  
UniProt Entry Name:GP1BA_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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