Human PDGFB ELISA Kit (HUEB0204)- High Sensitivity

Product Name:	Human Platelet-derived growth factor subunit B (PDGFB) ELISA Kit
Product Code:	HUEB0204
Alias:	Platelet-derived growth factor subunit B, PDGF subunit B, PDGF-2, Platelet-derived growth factor B chain, Platelet-derived growth factor beta polypeptide, Proto-oncogene c-Sis, PDGFB, PDGF2, SIS
Uniprot:	P01127
Reactivity:	Human
Range:	31.2-2000 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:	PDGFB: Growth factor that plays an essential role in the regulation of embryonic development, cell proliferation, cell migration, survival and chemotaxis. Potent mitogen for cells of mesenchymal origin. Required for normal proliferation and recruitment of pericytes and vascular smooth muscle cells in the central nervous system, skin, lung, heart and placenta. Required for normal blood vessel development, and for normal development of kidney glomeruli. Plays an important role in wound healing. Signaling is modulated by the formation of heterodimers with PDGFA. A chromosomal aberration involving PDGFB is found in dermatofibrosarcoma protuberans. Translocation t(17;22)(q22;q13) with PDGFB. Belongs to the PDGF/VEGF growth factor family.
UniProt Protein Details:	Protein type:Oncoprotein; Secreted, signal peptide; Motility/polarity/chemotaxis; Secreted Chromosomal Location of Human Ortholog: 22q13.1 Cellular Component: Golgi membrane; extracellular space; cell surface; basolateral plasma membrane; endoplasmic reticulum lumen; cytoplasm; extracellular region Molecular Function:collagen binding; identical protein binding; protein binding; protein homodimerization activity; growth factor activity; protein heterodimerization activity; platelet-derived growth factor binding; platelet-derived growth factor receptor binding; superoxide-generating NADPH oxidase activator activity; chemoattractant activity Biological Process: extracellular matrix organization and biogenesis; positive regulation of cyclin-dependent protein kinase activity; nerve growth factor receptor signaling pathway; positive regulation of transcription, DNA-dependent; heart development; positive regulation of fibroblast growth factor receptor signaling pathway; cell projection biogenesis; protein amino acid phosphorylation; positive regulation of MAP kinase activity; monocyte chemotaxis; positive regulation of fibroblast proliferation; positive chemotaxis; transforming growth factor beta receptor signaling pathway; cell growth; positive regulation of mitotic cell cycle, embryonic; response to drug; substrate-bound cell migration; platelet activation; fibroblast growth factor receptor signaling pathway; positive regulation of mitosis; positive regulation of chemotaxis; activation of protein kinase B; positive regulation of blood vessel endothelial cell migration; positive regulation of protein amino acid autophosphorylation; positive regulation of phosphoinositide 3-kinase cascade; positive regulation of peptidyl-tyrosine phosphorylation; activation of protein kinase activity; blood vessel morphogenesis; positive regulation of endothelial cell proliferation; negative regulation of transcription, DNA-dependent; actin cytoskeleton organization and biogenesis; embryonic placenta development; peptidyl-tyrosine phosphorylation; positive regulation of smooth muscle cell proliferation; platelet-derived growth factor receptor signaling pathway; positive regulation of smooth muscle cell migration; response to estradiol stimulus; response to insulin stimulus; platelet degranulation; positive regulation of MAPKKK cascade; positive regulation of glomerular filtration; positive regulation of cell proliferation; response to wounding; hemopoiesis; DNA replication; negative regulation of cell migration; epidermal growth factor receptor signaling pathway; phosphoinositide-mediated signaling; negative regulation of phosphatidylinositol biosynthetic process; positive regulation of phosphoinositide 3-kinase activity; peptidyl-serine phosphorylation; response to hypoxia; innate immune response; blood coagulation; positive regulation of DNA replication; positive regulation of cell migration Disease: Meningioma, Familial, Susceptibility To; Dermatofibrosarcoma Protuberans; Basal Ganglia Calcification, Idiopathic, 5; Basal Ganglia Calcification, Idiopathic, 1
NCBI Summary:	The protein encoded by this gene is a member of the platelet-derived growth factor family. The four members of this family are mitogenic factors for cells of mesenchymal origin and are characterized by a motif of eight cysteines. This gene product can exist either as a homodimer (PDGF-BB) or as a heterodimer with the platelet-derived growth factor alpha polypeptide (PDGF-AB), where the dimers are connected by disulfide bonds. Mutations in this gene are associated with meningioma. Reciprocal translocations between chromosomes 22 and 17, at sites where this gene and that for collagen type 1, alpha 1 are located, are associated with a particular type of skin tumor called dermatofibrosarcoma protuberans resulting from unregulated expression of growth factor. Two alternatively spliced transcript variants encoding different isoforms have been identified for this gene. [provided by RefSeq, Feb 2013]
UniProt Code:	P01127
NCBI GenInfo Identifier:	129724
NCBI Gene ID:	5155
NCBI Accession:	P01127.1
UniProt Secondary Accession:	P01127,P78431, Q15354, Q6FHE7, Q9UF23, G3XAG8,
UniProt Related Accession:	P01127
Molecular Weight:	241
NCBI Full Name:	Platelet-derived growth factor subunit B
NCBI Synonym Full Names:	platelet-derived growth factor beta polypeptide
NCBI Official Symbol:	PDGFB
NCBI Official Synonym Symbols:	SIS; SSV; IBGC5; PDGF2; c-sis; PDGF-2
NCBI Protein Information:	platelet-derived growth factor subunit B; becaplermin; PDGF, B chain; PDGF subunit B; proto-oncogene c-Sis; platelet-derived growth factor 2; platelet-derived growth factor B chain; platelet-derived growth factor, beta polypeptide (oncogene SIS); platelet-derived growth factor beta polypeptide (simian sarcoma viral (v-sis) oncogene homolog)
UniProt Protein Name:	Platelet-derived growth factor subunit B
UniProt Synonym Protein Names:	PDGF-2; Platelet-derived growth factor B chain; Platelet-derived growth factor beta polypeptide; Proto-oncogene c-Sis; INN: Becaplermin
Protein Family:	Platelet-derived growth factor
UniProt Gene Name:	PDGFB
UniProt Entry Name:	PDGFB_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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