Human EGR1 ELISA Kit (HUEB0650)- High Sensitivity

Product Name:	Human Early growth response protein 1 (EGR1) ELISA Kit
SKU:	HUEB0650
Size:	96T
Target:	Human Early growth response protein 1 (EGR1)
Synonyms:	AT225, Nerve growth factor-induced protein A, Transcription factor ETR103, Transcription factor Zif268, Zinc finger protein 225, Zinc finger protein Krox-24, NGFI-A, EGR-1, KROX24, ZNF225
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.156-10ng/mL
Sensitivity:	0.078 ng/mL

Intra CV:

4.9%

Inter CV:

9.1%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	106-116%	108-117%	103-113%	91-103%
EDTA Plasma(N=5)	109-119%	103-111%	97-109%	87-97%
Heparin Plasma(N=5)	103-113%	106-117%	97-106%	94-104%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	102	96-108
Plasma	104	98-110

Function:

Transcriptional regulator (PubMed:20121949). Recognizes and binds to the DNA sequence 5'-GCG(T/G)GGGCG-3'(EGR-site) in the promoter region of target genes (By similarity). Binds double-stranded target DNA, irrespective of the cytosine methylation status (PubMed:25258363, PubMed:25999311). Regulates the transcription of numerous target genes, and thereby plays an important role in regulating the response to growth factors, DNA damage, and ischemia. Plays a role in the regulation of cell survival, proliferation and cell death. Activates expression of p53/TP53 and TGFB1, and thereby helps prevent tumor formation. Required for normal progress through mitosis and normal proliferation of hepatocytes after partial hepatectomy. Mediates responses to ischemia and hypoxia; regulates the expression of proteins such as IL1B and CXCL2 that are involved in inflammatory processes and development of tissue damage after ischemia. Regulates biosynthesis of luteinizing hormone (LHB) in the pituitary.

Uniprot:	P18146
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Early growth response protein 1
Sub Unit:	Interacts with SNAI1 and SP1 upon 12-O-tetradecanoylphorbol-13-acetate (TPA) induction.
Research Area:	Neurosciences
Subcellular Location:	Nucleus Cytoplasm
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	EGR1: Transcriptional regulator. Recognizes and binds to the DNA sequence 5'-CGCCCCCGC-3'(EGR-site). Activates the transcription of target genes whose products are required for mitogenesis and differentiation. By growth factors. Belongs to the EGR C2H2-type zinc-finger protein family.
UniProt Protein Details:	Protein type:DNA-binding; C2H2-type zinc finger protein; Transcription factor Chromosomal Location of Human Ortholog: 5q31.1 Cellular Component: nucleoplasm; cytoplasm; nucleus Molecular Function:histone acetyltransferase binding; protein binding; DNA binding; sequence-specific DNA binding; metal ion binding; double-stranded DNA binding; transcription factor binding; transcription factor activity Biological Process: regulation of long-term neuronal synaptic plasticity; circadian rhythm; response to nutrient levels; transcription from RNA polymerase II promoter; cytokine and chemokine mediated signaling pathway; positive regulation of transcription, DNA-dependent; response to amphetamine; negative regulation of transcription from RNA polymerase II promoter; response to cocaine; BMP signaling pathway; response to ethanol; learning and/or memory; cellular response to insulin stimulus; positive regulation of neuron apoptosis; response to glucose stimulus; oligodendrocyte differentiation; positive regulation of transcription from RNA polymerase II promoter; response to electrical stimulus; T cell differentiation; regulation of protein sumoylation; negative regulation of apoptosis
NCBI Summary:	The protein encoded by this gene belongs to the EGR family of C2H2-type zinc-finger proteins. It is a nuclear protein and functions as a transcriptional regulator. The products of target genes it activates are required for differentitation and mitogenesis. Studies suggest this is a cancer suppressor gene. [provided by RefSeq, Dec 2014]
UniProt Code:	P18146
NCBI GenInfo Identifier:	119242
NCBI Gene ID:	1958
NCBI Accession:	P18146.1
UniProt Related Accession:	P18146
Molecular Weight:	543
NCBI Full Name:	Early growth response protein 1
NCBI Synonym Full Names:	early growth response 1
NCBI Official Symbol:	EGR1
NCBI Official Synonym Symbols:	TIS8; AT225; G0S30; NGFI-A; ZNF225; KROX-24; ZIF-268
NCBI Protein Information:	early growth response protein 1; EGR-1; zinc finger protein 225; transcription factor ETR103; transcription factor Zif268; zinc finger protein Krox-24; nerve growth factor-induced protein A
UniProt Protein Name:	Early growth response protein 1
UniProt Synonym Protein Names:	AT225; Nerve growth factor-induced protein A; NGFI-A; Transcription factor ETR103; Transcription factor Zif268; Zinc finger protein 225; Zinc finger protein Krox-24
Protein Family:	Early growth response protein
UniProt Gene Name:	EGR1
UniProt Entry Name:	EGR1_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.

ELISA	Antibodies
Human EGR1 / Early growth response protein 1 ELISA Kit	Anti-EGR1 Antibody (CAB2722)
Human EGR1 (Early Growth Response Protein 1) CLIA Kit (HUES01185)	Anti-EGR1 Antibody (CAB7266)
EGR1/2 Transcription Factor Activity Assay	EEF1G Antibody (PACO14369)
	EGLN2 Antibody, Biotin conjugated (PACO26412)
	EGR1 Antibody (PACO26413)