Human Immunology ELISA Kits 7

Human Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta isoform (PIK3CD) ELISA Kit

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SKU:
HUEB1520
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
O00329
Range:
0.312-20 ng/mL
ELISA Type:
Sandwich
Synonyms:
PIK3CD, Phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit delta isoform
Reactivity:
Human

Description

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Product Name:Human Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta isoform (PIK3CD) ELISA Kit
Product Code:HUEB1520
Alias:Phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit delta isoform, PI3-kinase subunit delta, PI3K-delta, PI3Kdelta, PtdIns-3-kinase subunit delta, Phosphatidylinositol-4, 5-bisphosphate 3-kinase 110 kDa catalytic subunit delta, PtdIns-3-kinase subunit p110-delta, p110delta, PIK3CD, 2.7.1.153
Uniprot:O00329
Reactivity:Human
Range:0.312-20 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:PIK3CD: Phosphoinositide-3-kinase (PI3K) that phosphorylates PftdIns(4,5)P2 (Phosphatidylinositol 4,5-bisphosphate) to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3). PIP3 plays a key role by recruiting PH domain-containing proteins to the membrane, including AKT1 and PDPK1, activating signaling cascades involved in cell growth, survival, proliferation, motility and morphology. Mediates immune responses. Plays a role in B-cell development, proliferation, migration, and function. Required for B-cell receptor (BCR) signaling. Mediates B-cell proliferation response to anti-IgM, anti-CD40 and IL4 stimulation. Promotes cytokine production in response to TLR4 and TLR9. Required for antibody class switch mediated by TLR9. Involved in the antigen presentation function of B-cells. Involved in B-cell chemotaxis in response to CXCL13 and sphingosine 1-phosphate (S1P). Required for proliferation, signaling and cytokine production of naive, effector and memory T-cells. Required for T-cell receptor (TCR) signaling. Mediates TCR signaling events at the immune synapse. Activation by TCR leads to antigen-dependent memory T-cell migration and retention to antigenic tissues. Together with PIK3CG participates in T-cell development. Contributes to T-helper cell expansion and differentiation. Required for T-cell migration mediated by homing receptors SELL/CD62L, CCR7 and S1PR1 and antigen dependent recruitment of T-cells. Together with PIK3CG is involved in natural killer (NK) cell development and migration towards the sites of inflammation. Participates in NK cell receptor activation. Have a role in NK cell maturation and cytokine production. Together with PIK3CG is involved in neutrophil chemotaxis and extravasation. Together with PIK3CG participates in neutrophil respiratory burst. Have important roles in mast-cell development and mast cell mediated allergic response. Involved in stem cell factor (SCF)-mediated proliferation, adhesion and migration. Required for allergen-IgE-induced degranulation and cytokine release. The lipid kinase activity is required for its biological function. Isoform 2 may be involved in stabilizing total RAS levels, resulting in increased ERK phosphorylation and increased PI3K activity. Belongs to the PI3/PI4-kinase family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Carbohydrate Metabolism - inositol phosphate; Kinase, lipid; EC 2.7.1.153; Motility/polarity/chemotaxis

Chromosomal Location of Human Ortholog: 1p36.2

Cellular Component: mast cell granule; plasma membrane; cytosol; phosphoinositide 3-kinase complex

Molecular Function:protein binding; phosphatidylinositol-4,5-bisphosphate 3-kinase activity; 1-phosphatidylinositol-3-kinase activity; ATP binding; phosphoinositide 3-kinase activity; phosphatidylinositol-4-phosphate 3-kinase activity

Biological Process: respiratory burst during defense response; phosphoinositide 3-kinase cascade; T cell activation; nerve growth factor receptor signaling pathway; natural killer cell activation; signal transduction; T cell receptor signaling pathway; protein amino acid phosphorylation; B cell receptor signaling pathway; B cell homeostasis; phosphoinositide phosphorylation; natural killer cell differentiation; phospholipid metabolic process; inflammatory response; T cell differentiation; epidermal growth factor receptor signaling pathway; neutrophil chemotaxis; adaptive immune response; phosphoinositide-mediated signaling; fibroblast growth factor receptor signaling pathway; B cell activation; mast cell chemotaxis; cytokine production; mast cell degranulation; phosphatidylinositol biosynthetic process; innate immune response

Disease: Immunodeficiency 14

NCBI Summary:Phosphoinositide 3-kinases (PI3Ks) phosphorylate inositol lipids and are involved in the immune response. The protein encoded by this gene is a class I PI3K found primarily in leukocytes. Like other class I PI3Ks (p110-alpha p110-beta, and p110-gamma), the encoded protein binds p85 adapter proteins and GTP-bound RAS. However, unlike the other class I PI3Ks, this protein phosphorylates itself, not p85 protein.[provided by RefSeq, Jul 2010]
UniProt Code:O00329
NCBI GenInfo Identifier:67477424
NCBI Gene ID:5293
NCBI Accession:O00329.2
UniProt Secondary Accession:O00329,O15445, Q5SR49, A6NCG0, G1FFP1,
UniProt Related Accession:O00329
Molecular Weight:119,479 Da
NCBI Full Name:Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform
NCBI Synonym Full Names:phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit delta
NCBI Official Symbol:PIK3CD  
NCBI Official Synonym Symbols:APDS; PI3K; IMD14; p110D; P110DELTA  
NCBI Protein Information:phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform; PI3Kdelta; phosphoinositide-3-kinase C; PI3-kinase p110 subunit delta; ptdIns-3-kinase subunit p110-delta; phosphoinositide-3-kinase, catalytic, delta polypeptide variant p37d
UniProt Protein Name:Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform
UniProt Synonym Protein Names:Phosphatidylinositol 4,5-bisphosphate 3-kinase 110 kDa catalytic subunit delta
UniProt Gene Name:PIK3CD  
UniProt Entry Name:PK3CD_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.
ELISA
Human PIK3CD ELISA Kit
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