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Rat Podoplanin (Pdpn) ELISA Kit (RTEB1364)

SKU:
RTEB1364
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q64294
ELISA Type:
Sandwich
Synonyms:
PDPN, Aggrus, Gp38
Reactivity:
Rat
€649
Frequently bought together:

Description

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Rat Podoplanin (Pdpn) ELISA Kit

The Rat Podoplanin (PDPN) ELISA Kit is a reliable and accurate tool for detecting levels of podoplanin in rat serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, guaranteeing precise and consistent results for a variety of research studies.Podoplanin, also known as PDPN, is a protein that plays a crucial role in cell adhesion and migration, especially in the formation of lymphatic vessels. Dysregulation of podoplanin has been implicated in various diseases, including cancer and inflammatory disorders.

Therefore, this ELISA kit is essential for studying the role of podoplanin in disease development and progression, as well as for the development of potential therapeutic strategies.With its easy-to-use format and reliable performance, the Rat Podoplanin (PDPN) ELISA Kit is a valuable tool for researchers looking to investigate the function and importance of podoplanin in a rat model system.

Product Name:Rat Podoplanin (Pdpn) ELISA Kit
SKU:RTEB1364
Size:96T
Target:Rat Podoplanin (Pdpn)
Synonyms:E11 antigen epitope, RTI140, T1-alpha, Type I cell 40 kDa protein, T1A
Assay Type:Sandwich
Detection Method:ELISA
Reactivity:Rat
Detection Range:0.312-20ng/mL
Sensitivity:0.162ng/mL
Intra CV:Provided with the Kit
Inter CV:Provided with the Kit
Linearity:Provided with the Kit
Recovery:Provided with the Kit
Function:Mediates effects on cell migration and adhesion through its different partners. During development plays a role in blood and lymphatic vessels separation by binding CLEC1B, triggering CLEC1B activation in platelets and leading to platelet activation and/or aggregation. Interaction with CD9, on the contrary, attenuates platelet aggregation and pulmonary metastasis induced by PDPN. Mediates effects on cell migration and adhesion through its different partners. Through MSN or EZR interaction promotes epithelial-mesenchymal transition (EMT) leading to ERZ phosphorylation and triggering RHOA activation leading to cell migration increase and invasiveness. Interaction with CD44 promotes directional cell migration in epithelial and tumor cells (By similarity). In lymph nodes (LNs), controls fibroblastic reticular cells (FRCs) adhesion to the extracellular matrix (ECM) and contraction of the actomyosin by maintaining ERM proteins (EZR; MSN and RDX) and MYL9 activation through association with unknown transmembrane proteins. Engagement of CLEC1B by PDPN promotes FRCs relaxation by blocking lateral membrane interactions leading to reduction of ERM proteins (EZR; MSN and RDX) and MYL9 activation (By similarity). Through binding with LGALS8 may participate to connection of the lymphatic endothelium to the surrounding extracellular matrix. In keratinocytes, induces changes in cell morphology showing an elongated shape, numerous membrane protrusions, major reorganization of the actin cytoskeleton, increased motility and decreased cell adhesion. Controls invadopodia stability and maturation leading to efficient degradation of the extracellular matrix (ECM) in tumor cells through modulation of RHOC activity in order to activate ROCK1/ROCK2 and LIMK1/LIMK2 and inactivation of CFL1 (By similarity). Required for normal lung cell proliferation and alveolus formation at birth (By similarity). Does not function as a water channel or as a regulator of aquaporin-type water channels (By similarity). Does not have any effect on folic acid or amino acid transport (By similarity).
Uniprot:Q64294
Sample Type:Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:Natural and recombinant rat Podoplanin
Sub Unit:Homodimer. Interacts with CLEC1B; the interaction is independent of CLEC1B glycosylation and activates CLEC1B; the interaction is dependent of sialic acid on O-glycans. Interacts with CD9; this interaction is homophilic and attenuates platelet aggregation and pulmonary metastasis induced by PDPN. Interacts with LGALS8; the interaction is glycosylation-dependent; may participate to connection of the lymphatic endothelium to the surrounding extracellular matrix. Interacts with HSPA9. Interacts (via extracellular domain) with CD44; this interaction is required for PDPN-mediated directional migration and regulation of lamellipodia extension/stabilization during cell spreading and migration. Interacts (via cytoplasmic domain) with MSN and EZR; activates RHOA and promotes epithelial-mesenchymal transition. Interacts with CCL21; relocalized PDPN to the basolateral membrane.
Research Area:Cardiovascular
Subcellular Location:Membrane Single-pass type I membrane protein Cell projection Lamellipodium membrane Single-pass type I membrane protein Cell projection Filopodium membrane Single-pass type I membrane protein Cell projection Microvillus membrane Single-pass type I membrane protein Cell projection Ruffle membrane Single-pass type I membrane protein Membrane raft Apical cell membrane Basolateral cell membrane Cell projection Invadopodium Localized to actin-rich microvilli and plasma membrane projections such as filopodia, lamellipodia and ruffles (PubMed:9327748). Association to the lipid rafts is required for PDPN-induced epithelial to mesenchymal transition (EMT). Colocalizes with CD9 in tetraspanin microdomains. Localized at invadopodium adhesion rings in tumor cell. Association to the lipid rafts is essential for PDPN recruitment to invadopodia and ECM degradation (By similarity).
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:May be involved in cell migration and/or actin cytoskeleton organization. When expressed in keratinocytes, induces changes in cell morphology with transfected cells showing an elongated shape, numerous membrane protrusions, major reorganization of the actin cytoskeleton, increased motility and decreased cell adhesion. Required for normal lung cell proliferation and alveolus formation at birth. Induces platelet aggregation. Does not have any effect on folic acid or amino acid transport. Does not function as a water channel or as a regulator of aquaporin-type water channels.
NCBI Summary:membrane glycoprotein that may function in lung development [RGD, Feb 2006]
UniProt Code:Q64294
NCBI GenInfo Identifier:2498421
NCBI Gene ID:54320
NCBI Accession:Q64294.1
UniProt Secondary Accession:Q64294,O08731, O55210,
UniProt Related Accession:Q64294
Molecular Weight:17,579 Da
NCBI Full Name:Podoplanin
NCBI Synonym Full Names:podoplanin
NCBI Official Symbol:Pdpn
NCBI Official Synonym Symbols:E11; Gp38; OTS-8; RTI40; T1-alpha
NCBI Protein Information:podoplanin; T1A; RTI140; glycoprotein 38; E11 antigen epitope; type I cell 40 kDa protein; epithelial cell surface transmembrane protein antigen; pulmonary type I alveolar epithelial cell transmembrane differentiation marker
UniProt Protein Name:Podoplanin
UniProt Synonym Protein Names:E11 antigen epitope; Glycoprotein 38; Gp38; OTS-8; RTI140; T1-alpha; T1A; Type I cell 40 kDa protein
Protein Family:Podoplanin
UniProt Gene Name:Pdpn
UniProt Entry Name:PDPN_RAT
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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