Rat Proto-oncogene tyrosine-protein kinase receptor Ret (Ret) ELISA Kit
The Rat Proto-Oncogene Tyrosine-Protein Kinase Receptor RET (RET) ELISA Kit is a valuable tool for the detection of RET levels in rat serum, plasma, and cell culture supernatants. This kit is known for its high sensitivity and specificity, ensuring accurate and reliable results for various research applications.The RET protein is a key player in cell signalling and growth regulation, particularly in the development of neural crest cells and the formation of enteric nervous system.
Dysregulation of RET has been implicated in a number of diseases, including thyroid cancer and multiple endocrine neoplasia type 2 (MEN2), making it a critical biomarker for understanding these conditions and exploring potential therapeutic interventions.Overall, the Rat Proto-Oncogene Tyrosine-Protein Kinase Receptor RET (RET) ELISA Kit is an essential tool for researchers studying RET signalling pathways and its implications in disease development.
Product Name:
Rat Proto-oncogene tyrosine-protein kinase receptor Ret (Ret) ELISA Kit
SKU:
RTEB1073
Size:
96T
Target:
Rat Proto-oncogene tyrosine-protein kinase receptor Ret (Ret)
Synonyms:
Proto-oncogene tyrosine-protein kinase receptor Ret, 2.7.10.1, Ret
Assay Type:
Sandwich
Detection Method:
ELISA
Reactivity:
Rat
Detection Range:
0.156-10ng/ml
Sensitivity:
0.09ng/mL
Intra CV:
4.6%
Inter CV:
7.7%
Linearity:
Sample
1:2
1:4
1:8
1:16
Serum(N=5)
108-118%
110-120%
106-118%
102-111%
EDTA Plasma(N=5)
102-112%
106-117%
100-112%
84-93%
Heparin Plasma(N=5)
95-105%
81-93%
88-98%
88-98%
Recovery:
Sample Type
Average(%)
Recovery Range(%)
Serum
87
81-93
Plasma
89
83-95
Function:
Receptor tyrosine-protein kinase involved in numerous cellular mechanisms including cell proliferation, neuronal navigation, cell migration, and cell differentiation upon binding with glial cell derived neurotrophic factor family ligands. Phosphorylates PTK2/FAK1. Regulates both cell death/survival balance and positional information. Required for the molecular mechanisms orchestration during intestine organogenesis; involved in the development of enteric nervous system and renal organogenesis during embryonic life, and promotes the formation of Peyer's patch-like structures, a major component of the gut-associated lymphoid tissue. Modulates cell adhesion via its cleavage by caspase in sympathetic neurons and mediates cell migration in an integrin (e.g. ITGB1 and ITGB3)-dependent manner. Involved in the development of the neural crest. Active in the absence of ligand, triggering apoptosis through a mechanism that requires receptor intracellular caspase cleavage. Acts as a dependence receptor; in the presence of the ligand GDNF in somatotrophs (within pituitary), promotes survival and down regulates growth hormone (GH) production, but triggers apoptosis in absence of GDNF. Regulates nociceptor survival and size. Triggers the differentiation of rapidly adapting (RA) mechanoreceptors. Mediator of several diseases such as neuroendocrine cancers; these diseases are characterized by aberrant integrins-regulated cell migration.
Uniprot:
G3V9H8
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant rat Proto-oncogene tyrosine-protein kinase receptor Ret
Sub Unit:
Phosphorylated form interacts with the PBT domain of DOK2, DOK4 and DOK5. The phosphorylated form interacts with PLCG1 and GRB7. Interacts (not phosphorylated) with PTK2/FAK1 (via FERM domain). Extracellular cell-membrane anchored RET cadherin fragments form complex in neurons with reduced trophic status, preferentially at the contact sites between somas. Interacts with AIP in the pituitary gland; this interaction prevents the formation of the AIP-survivin complex. Binds to ARTN. Interacts (inactive) with CBLC and CD2AP; dissociates upon activation by GDNF which increases CBLC:CD2AP interaction.
Research Area:
Cancer
Subcellular Location:
Cell membrane Single-pass type I membrane protein Endosome membrane Single-pass type I membrane protein
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
Ret: a proto-oncogenic receptor tyrosine kinase. Receptor for glial cell line-derived neurotropic factor (GDNF) and its congeners neurturin, persephin and artemin. Part of a multicompetent receptor complex with other membrane-bound ligand-binding GDNF family receptors (aGFRs). Required for development of the kidney and neural crest-derived cell types. Three alternatively spliced isoforms have been described.Protein type: Oncoprotein; Protein kinase, tyrosine (receptor); EC 2.7.10.1; Kinase, protein; Protein kinase, TK; Membrane protein, integral; TK group; Ret familyCellular Component: intracellular membrane-bound organelle; integral to plasma membrane; cytoplasm; plasma membrane; endosome membrane; intracellular; receptor complex; lipid raftMolecular Function: transmembrane receptor protein tyrosine kinase activity; calcium ion binding; ATP bindingBiological Process: regulation of cell adhesion; caspase activation; response to drug; nervous system development; positive regulation of neuron maturation; peptidyl-tyrosine phosphorylation; positive regulation of cell size; membrane protein proteolysis; positive regulation of transcription, DNA-dependent; MAPKKK cascade; positive regulation of cell adhesion mediated by integrin; response to pain; regulation of axonogenesis; neuron maturation; enteric nervous system development; neuron differentiation; retina development in camera-type eye; ureteric bud development; embryonic epithelial tube formation; neuron adhesion; homophilic cell adhesion; neural crest cell migration; transmembrane receptor protein tyrosine kinase signaling pathway; positive regulation of cell migration
UniProt Protein Details:
NCBI Summary:
may play a role in excretory system and enteric nervous system development; human homolog is associated with Hirschsprung disease and Multiple Endocrine Neoplasia type 2 [RGD, Feb 2006]
proto-oncogene tyrosine-protein kinase receptor Ret isoform a
NCBI Synonym Full Names:
ret proto-oncogene
NCBI Official Symbol:
Ret
NCBI Official Synonym Symbols:
NCBI Protein Information:
proto-oncogene tyrosine-protein kinase receptor Ret
UniProt Protein Name:
Proto-oncogene tyrosine-protein kinase receptor Ret
UniProt Synonym Protein Names:
Protein Family:
Retbindin
UniProt Gene Name:
Ret
UniProt Entry Name:
RET_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.
