Product Name:	Human DLG4/PSD95 ELISA Kit
Product Code:	HUFI01144
Size:	96 Assays
Alias:	DLG4, Disks large homolog 4, Postsynaptic density protein 95, PSD-95, Synapse-associated protein 90, SAP-90, SAP90
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human DLG4 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.188ng/ml
Range:	0.313-20ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human DLG4 and the recovery rates were calculated by comparing the measured value to the expected amount of Human DLG4 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	86-105	99
EDTA plasma(n=5)	86-99	94
UFH plasma(n=5)	90-104	96

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human DLG4 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	85-101%	92-100%	85-101%
EDTA plasma(n=5)	82-95%	87-99%	82-95%
UFH plasma(n=5)	80-100%	85-98%	84-95%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	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

Uniprot

P78352

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.
UniProt Protein Details:	Protein type:Adaptor/scaffold Chromosomal Location of Human Ortholog: 17p13.1 Cellular Component: cortical cytoskeleton; dendrite cytoplasm; voltage-gated potassium channel complex; basolateral plasma membrane; endoplasmic reticulum; postsynaptic density; dendritic spine; excitatory synapse; postsynaptic membrane; synaptic vesicle; extrinsic to internal side of plasma membrane; cytoplasm; plasma membrane; ionotropic glutamate receptor complex; synapse; cell junction Molecular Function:P2Y1 nucleotide receptor binding; protein C-terminus binding; guanylate kinase activity; ionotropic glutamate receptor binding; protein binding; beta-1 adrenergic receptor binding; acetylcholine receptor binding; protein complex binding; D1 dopamine receptor binding; protein phosphatase binding; PDZ domain binding Biological Process: regulation of long-term neuronal synaptic plasticity; axon guidance; nervous system development; synaptic vesicle maturation; social behavior; learning; signal transduction; positive regulation of synaptic transmission; establishment and/or maintenance of epithelial cell polarity; synaptic transmission; elevation of cytosolic calcium ion concentration; establishment of protein localization; protein complex assembly; neuromuscular process controlling balance; nucleotide phosphorylation; negative regulation of receptor internalization
NCBI Summary:	This gene encodes a member of the membrane-associated guanylate kinase (MAGUK) family. It heteromultimerizes with another MAGUK protein, DLG2, and is recruited into NMDA receptor and potassium channel clusters. These two MAGUK proteins may interact at postsynaptic sites to form a multimeric scaffold for the clustering of receptors, ion channels, and associated signaling proteins. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P78352
NCBI GenInfo Identifier:	71658825
NCBI Gene ID:	1742
NCBI Accession:	P78352.3
UniProt Secondary Accession:	P78352,Q92941, Q9UKK8, B7Z1S1, G5E939,
UniProt Related Accession:	P78352
Molecular Weight:	80,125 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:	PSD95; SAP90; SAP-90
NCBI Protein Information:	disks large homolog 4; discs large homolog 4; Tax interaction protein 15; synapse-associated protein 90; postsynaptic density protein 95; post-synaptic density protein 95
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:	Disks large
UniProt Gene Name:	DLG4
UniProt Entry Name:	DLG4_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

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.