Mouse Cell Signalling ELISA Kits 3

Mouse Disks large homolog 4 (Dlg4) ELISA Kit

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SKU:
MOEB1060

Description

system_update_altDatasheetsystem_update_altMSDS
Product Name:Mouse Disks large homolog 4 (Dlg4) ELISA Kit
Product Code:MOEB1060
Alias:Disks large homolog 4, Postsynaptic density protein 95, PSD-95, Synapse-associated protein 90, SAP-90, SAP90, Dlg4, Dlgh4, Psd95
Uniprot:Q62108
Reactivity:Mouse
Range:Please contact us for more information
Detection Method:Sandwich
Size:96 Assay
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:PSD-95: a membrane associated guanylate kinase (MAGUK) scaffolding protein located in neural postsynaptic densities. Associates with NMDA receptor NR2 subunits via glutamate serine (aspartate/glutamate) valine motifs in the cytoplasmic tail of NMDA receptor subunits and shaker-type potassium channels. Required for synaptic plasticity associated with NMDA receptor signaling. Its overexpression or depletion changes the ratio of excitatory to inhibitory synapses in hippocampal neurons. High levels in postsynaptic density of neurons in the forebrain. Also in presynaptic region of inhibitory synapses formed by cerebellar basket cells on axon hillocks of Purkinje cells. May reduce the amplitude of ACCN3 acid-evoked currents by retaining the channel intracellularly. Binds tissue-type plasminogen activator (tPA), mediating neural NMDA gating via a complex of LRP1, PSD-95 and NMDAR. Interacts through its first two PDZ domains with NMDAR2A, NMDAR2B, NMDAR2C, NMDAR2D, ACCN3, certain splice forms of NMDAR1, KV4.2, CXADR and synGAP. Interacts through its first two PDZ domains with Kv1.1, Kv1.2, Kv1.3, Kv1.4 and HER4. Interacts through its first PDZ domain with GluR6 and Kv1.4. Interacts through its second PDZ domain with the PDZ domain of nNOS or the C-terminus of NOS1AP. May interact with 5-HT(2A). Interacts through its third PDZ domain with NLGN1, and probably with NLGN2 and NLGN3. Interacts through its guanylate kinase-like domain with SAPAP1, SAPAP2, SAPAP3, SAPAP4, MAP1A, and BEGAIN. Interacts through its guanylate kinase-like domain with KIF13B. Two alternatively spliced human isoforms have been reported. Palmitoylation of isoform 1 is required for targeting to postsynaptic density.Protein type: Adaptor/scaffoldCellular Component: basolateral plasma membrane; cell junction; cell projection; cortical cytoskeleton; cytoplasm; cytosol; dendrite; dendrite cytoplasm; dendritic spine; endoplasmic reticulum; excitatory synapse; extrinsic to internal side of plasma membrane; ionotropic glutamate receptor complex; membrane; plasma membrane; postsynaptic density; postsynaptic membrane; synapse; synaptic vesicle; voltage-gated potassium channel complexMolecular Function: acetylcholine receptor binding; beta-1 adrenergic receptor binding; D1 dopamine receptor binding; glutamate receptor binding; guanylate kinase activity; ionotropic glutamate receptor binding; kinase binding; neurexin binding; P2Y1 nucleotide receptor binding; PDZ domain binding; protein binding; protein C-terminus binding; protein complex binding; protein kinase binding; protein phosphatase binding; receptor binding; structural molecule activityBiological Process: elevation of cytosolic calcium ion concentration; establishment and/or maintenance of epithelial cell polarity; establishment of protein localization; locomotory behavior; negative regulation of receptor internalization; neuromuscular process controlling balance; positive regulation of synaptic transmission; protein complex assembly; receptor clustering; regulation of long-term neuronal synaptic plasticity; regulation of neuronal synaptic plasticity; response to cocaine; social behavior; synaptic transmission; synaptic vesicle maturation
UniProt Protein Details:
NCBI Summary:
UniProt Code:Q62108
NCBI GenInfo Identifier:2497501
NCBI Gene ID:13385
NCBI Accession:Q62108.1
UniProt Secondary Accession:Q62108,Q5NCV5, Q5NCV6, Q5NCV7, Q91WJ1
UniProt Related Accession:Q62108
Molecular Weight:80,102 Da
NCBI Full Name:Disks large homolog 4
NCBI Synonym Full Names:discs, large homolog 4 (Drosophila)
NCBI Official Symbol:Dlg4  
NCBI Official Synonym Symbols:Dlgh4; PSD95; SAP90; PSD-95; SAP90A  
NCBI Protein Information:disks large homolog 4
UniProt Protein Name:Disks large homolog 4
UniProt Synonym Protein Names:Postsynaptic density protein 95; PSD-95; Synapse-associated protein 90; SAP-90; SAP90
Protein Family:
UniProt Gene Name:Dlg4  
UniProt Entry Name:DLG4_MOUSE
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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Additional Information

Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q62108
ELISA Type:
Sandwich
Reactivity:
Mouse
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