Human Serum response factor (SRF) ELISA Kit

Product Name:	Human Serum response factor (SRF) ELISA Kit
SKU:	HUEB2500
Size:	96T
Target:	Human Serum response factor (SRF)
Synonyms:	SRF
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	31.2-2000pg/mL
Sensitivity:	13pg/mL

Intra CV:

4.7%

Inter CV:

8.1%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	81-90%	84-93%	92-102%	99-109%
EDTA Plasma(N=5)	108-120%	111-121%	105-114%	95-105%
Heparin Plasma(N=5)	104-113%	88-96%	104-114%	100-112%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	87	81-93
Plasma	89	83-95

Function:

SRF is a transcription factor that binds to the serum response element (SRE), a short sequence of dyad symmetry located 300 bp to the 5' of the site of transcription initiation of some genes (such as FOS). Required for cardiac differentiation and maturation.

Uniprot:	P11831
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Serum response factor
Sub Unit:	Binds DNA as a multimer, probably a dimer. Interacts with MLLT7/FOXO4, NKX3A and SSRP1. Interacts with ARID2 and SRFBP1 (By similarity). Forms complexes with ARID2, MYOCD, NKX2-5 and SRFBP1 (By similarity). Forms a nuclear ternary complex with MKL1 and SCAI (PubMed:19350017). Interacts with LPXN (PubMed:18497331). Interacts with OLFM2; the interaction promotes dissociation of SRF from the transcriptional repressor HEY2, facilitates binding of SRF to target genes and promotes smooth muscle differentiation (PubMed:25298399).
Research Area:	Cancer
Subcellular Location:	Nucleus
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	SRF: a transcription factor of the MADS domain family that binds to the serum response element (SRE). Regulates the transcription of immediate early genes including c-fos. Binds DNA as a multimer, probably a dimer.
UniProt Protein Details:	Protein type:Transcription factor; Motility/polarity/chemotaxis Chromosomal Location of Human Ortholog: 6p21.1 Cellular Component: cytoplasm; nuclear chromatin; nucleoplasm; nucleus Molecular Function:chromatin DNA binding; histone deacetylase binding; protein binding; protein homodimerization activity; RNA polymerase II transcription factor activity, enhancer binding; transcription factor activity; transcription factor binding Biological Process: angiogenesis involved in wound healing; associative learning; cardiac myofibril assembly; cell migration during sprouting angiogenesis; cell-matrix adhesion; developmental growth; erythrocyte development; heart development; heart looping; hippocampus development; long-term memory; mesoderm formation; morphogenesis of an epithelial sheet; mRNA transcription from RNA polymerase II promoter; muscle maintenance; negative regulation of cell migration; negative regulation of cell proliferation; neurite development; neuron development; neuron migration; patterning of blood vessels; platelet activation; platelet formation; positive regulation of axon extension; positive regulation of cell differentiation; positive regulation of filopodium formation; positive regulation of smooth muscle contraction; positive regulation of transcription by glucose; positive regulation of transcription factor activity; positive regulation of transcription from RNA polymerase II promoter; positive thymic T cell selection; regulation of cell adhesion; regulation of smooth muscle cell differentiation; regulation of water loss via skin; response to cytokine stimulus; response to hormone stimulus; response to hypoxia; response to toxin; sarcomere organization; skin morphogenesis; small GTPase mediated signal transduction; stress fiber formation; tangential migration from the subventricular zone to the olfactory bulb; thymus development; thyroid gland development; transcription from RNA polymerase II promoter; trophectodermal cell differentiation
NCBI Summary:	This gene encodes a ubiquitous nuclear protein that stimulates both cell proliferation and differentiation. It is a member of the MADS (MCM1, Agamous, Deficiens, and SRF) box superfamily of transcription factors. This protein binds to the serum response element (SRE) in the promoter region of target genes. This protein regulates the activity of many immediate-early genes, for example c-fos, and thereby participates in cell cycle regulation, apoptosis, cell growth, and cell differentiation. This gene is the downstream target of many pathways; for example, the mitogen-activated protein kinase pathway (MAPK) that acts through the ternary complex factors (TCFs). Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, May 2014]
UniProt Code:	P11831
NCBI GenInfo Identifier:	134876
NCBI Gene ID:	6722
NCBI Accession:	P11831.1
UniProt Secondary Accession:	P11831,Q5T648,
UniProt Related Accession:	P11831
Molecular Weight:	51,593 Da
NCBI Full Name:	Serum response factor
NCBI Synonym Full Names:	serum response factor
NCBI Official Symbol:	SRF
NCBI Official Synonym Symbols:	MCM1
NCBI Protein Information:	serum response factor
UniProt Protein Name:	Serum response factor
Protein Family:	Serum response factor
UniProt Gene Name:	SRF
UniProt Entry Name:	SRF_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 Serum response factor / SRF ELISA Kit	Anti-SRF Antibody (CAB0409)
SRF Transcription Factor Activity Assay	Anti-SRF Antibody (CAB16718)
SRF (Phospho-Ser99) Colorimetric Cell-Based ELISA Kit	Anti-SRF Antibody (CAB7731)
SRF Colorimetric Cell-Based ELISA Kit	Anti-Phospho-SRF-S103 pAb Antibody (CABP0829)
	SRF Antibody (PACO01541)