Product Name:	Human TWIST1 (Twist-related protein 1) ELISA Kit
Product Code:	HUFI05887
Size:	96 Assays
Alias:	ACS3 ELISA Kit, bHLHa38 ELISA Kit, BPES2 ELISA Kit, BPES3 ELISA Kit, H twist ELISA Kit, SCS ELISA Kit, TWIST ELISA Kit, twist homolog 1 ELISA Kit, Drosophila ELISA Kit, Twist related protein 1 ELISA Kit, TWIST1 ELISA Kit
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human TWIST1 (Twist-related protein 1) concentrations in serum plasma and other biological fluids.
Sensitivity:	< 18.75pg/ml
Range:	31.25-2000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:	Matrices listed below were spiked with certain level of Human TWIST1 (Twist-related protein 1) and the recovery rates were calculated by comparing the measured value to the expected amount of Human TWIST1 (Twist-related protein 1) in samples. Enquire for more information.
Linearity:	The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human TWIST1 (Twist-related protein 1) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. Enquire for more information.
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 Protein Function:	TWIST1: Acts as a transcriptional regulator. Inhibits myogenesis by sequestrating E proteins, inhibiting trans-activation by MEF2, and inhibiting DNA-binding by MYOD1 through physical interaction. This interaction probably involves the basic domains of both proteins. Also represses expression of proinflammatory cytokines such as TNFA and IL1B. Regulates cranial suture patterning and fusion. Activates transcription as a heterodimer with E proteins. Regulates gene expression differentially, depending on dimer composition. Homodimers induce expression of FGFR2 and POSTN while heterodimers repress FGFR2 and POSTN expression and induce THBS1 expression. Heterodimerization is also required for osteoblast differentiation. Efficient DNA binding requires dimerization with another bHLH protein. Homodimer or heterodimer with E proteins such as TCF3. ID1 binds preferentially to TCF3 but does not interact efficiently with TWIST1 so ID1 levels control the amount of TCF3 available to dimerize with TWIST1 and thus determine the type of dimer formed. Subset of mesodermal cells.
UniProt Protein Details:	Protein type:DNA-binding; Transcription regulation; Inhibitor; Cell development/differentiation Chromosomal Location of Human Ortholog: 7p21.2 Cellular Component: nucleus Molecular Function:protein domain specific binding; protein binding; protein homodimerization activity; protein heterodimerization activity; bHLH transcription factor binding; transcription factor binding Biological Process: transcription from RNA polymerase II promoter; embryonic forelimb morphogenesis; negative regulation of skeletal muscle development; muscle development; rhythmic process; neuron migration; palate development; negative regulation of transcription from RNA polymerase II promoter; embryonic hindlimb morphogenesis; negative regulation of histone phosphorylation; negative regulation of histone acetylation; odontogenesis; negative regulation of DNA damage response, signal transduction by p53 class mediator; negative regulation of osteoblast differentiation; ossification; in utero embryonic development; negative regulation of transcription factor activity; outer ear morphogenesis; embryonic cranial skeleton morphogenesis; negative regulation of tumor necrosis factor production; positive regulation of tumor necrosis factor production; positive regulation of fatty acid beta-oxidation; osteoblast differentiation; positive regulation of angiogenesis; neural tube closure; positive regulation of transcription from RNA polymerase II promoter; embryonic digit morphogenesis; negative regulation of transcription, DNA-dependent; regulation of bone mineralization; positive regulation of epithelial cell proliferation; negative regulation of apoptosis; negative regulation of phosphoinositide 3-kinase cascade Disease: Robinow-sorauf Syndrome; Craniosynostosis 1; Saethre-chotzen Syndrome
NCBI Summary:	Basic helix-loop-helix (bHLH) transcription factors have been implicated in cell lineage determination and differentiation. The protein encoded by this gene is a bHLH transcription factor and shares similarity with another bHLH transcription factor, Dermo1. The strongest expression of this mRNA is in placental tissue; in adults, mesodermally derived tissues express this mRNA preferentially. Mutations in this gene have been found in patients with Saethre-Chotzen syndrome. [provided by RefSeq, Jul 2008]
UniProt Code:	Q15672
NCBI GenInfo Identifier:	2498009
NCBI Gene ID:	7291
NCBI Accession:	Q15672.1
UniProt Secondary Accession:	Q15672,Q92487, Q99804, A4D128,
UniProt Related Accession:	Q15672
Molecular Weight:	Calculated: 20 kdaObserved: 34 kDa
NCBI Full Name:	Twist-related protein 1
NCBI Synonym Full Names:	twist family bHLH transcription factor 1
NCBI Official Symbol:	TWIST1
NCBI Official Synonym Symbols:	CRS; CSO; SCS; ACS3; CRS1; BPES2; BPES3; TWIST; bHLHa38
NCBI Protein Information:	twist-related protein 1; H-twist; twist homolog 1; B-HLH DNA binding protein; TWIST homolog of drosophila; class A basic helix-loop-helix protein 38; twist basic helix-loop-helix transcription factor 1
UniProt Protein Name:	Twist-related protein 1
UniProt Synonym Protein Names:	Class A basic helix-loop-helix protein 38; bHLHa38; H-twist
Protein Family:	Twist-related protein
UniProt Gene Name:	TWIST1
UniProt Entry Name:	TWST1_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.

Antibodies

Anti-Twist Antibody (CAB15596)[KO Validated]

Anti-Twist Antibody (CAB3237)

Anti-Twist Antibody (CAB7314)

TWIST1 Antibody (PACO12995)

TWIST1 Antibody (PACO18470)