HSF1 (Phospho-Ser121)Colorimetric Cell-Based ELISA Kit (CBCAB01631)

Product Name:	HSF1 (Phospho-Ser121)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01631
ELISA Type:	Cell-Based
Target:	HSF1 (Phospho-Ser121)
Reactivity:	Human, Mouse
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The HSF1 (Phospho-Ser121) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect HSF1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated HSF1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on HSF1 phosphorylation.

Qualitative determination of HSF1 (Phospho-Ser121) concentration is achieved by an indirect ELISA format. In essence, HSF1 (Phospho-Ser121) is captured by HSF1 (Phospho-Ser121)-specific primary (1Ãƒ¸) antibodies while the HRP-conjugated secondary (2Ãƒ¸) antibodies bind the Fc region of the 1Ãƒ¸ antibody. Through this binding, the HRP enzyme conjugated to the 2Ãƒ¸ antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1.	A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2.	Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.

Database Information:	Gene ID: 3297, UniProt ID: Q00613, OMIM: 140580, Unigene: Hs.530227/Hs.571397
Gene Symbol:	HSF1
Sub Type:	Phospho

UniProt Protein Function:	HSF1: a transcription factor that specifically binds heat shock promoter elements (HSE) and activates transcription. Induced in response to heat, heavy metals, and oxidative stress. In higher eukaryotes, HSF is unable to bind to HSEs unless the cells are stressed. Becomes phosphorylated in response to stress, forming homotrimers that bind DNA and activate transcription. Phosphorylation by PLK1 enhances nuclear translocation, and phosphorylation by CaMKII enhances transactivation. Phosphorylation by GSK3 and ERK1 induces binding by 14-3-3 and sequestration in the cytoplasm. In addition, during attenuation from the heat shock response, HSF1 is repressed by direct binding of Hsp70, HSP40, and HSF binding protein 1 (HSBP1). Four alternatively spliced isoforms have been described.
UniProt Protein Details:	Protein type:Transcription factor; DNA-binding Chromosomal Location of Human Ortholog: 8q24.3 Cellular Component: nucleoplasm; protein complex; pronucleus; cytoplasm; cytosol Molecular Function:protein binding; chromatin binding; transcription factor activity Biological Process: negative regulation of cell proliferation; embryonic placenta development; embryonic process involved in female pregnancy; mRNA transcription; female meiosis; positive regulation of transcription from RNA polymerase II promoter; response to lipopolysaccharide; defense response; spermatogenesis; negative regulation of tumor necrosis factor production; negative regulation of transcription from RNA polymerase II promoter; positive regulation of multicellular organism growth; protein amino acid phosphorylation
NCBI Summary:	The product of this gene is a heat-shock transcription factor. Transcription of heat-shock genes is rapidly induced after temperature stress. Hsp90, by itself and/or associated with multichaperone complexes, is a major repressor of this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	Q00613
NCBI GenInfo Identifier:	462333
NCBI Gene ID:	3297
NCBI Accession:	Q00613.1
UniProt Secondary Accession:	Q00613,Q53XT4, A8K4L0, A8MW26,
UniProt Related Accession:	Q00613
Molecular Weight:	529
NCBI Full Name:	Heat shock factor protein 1
NCBI Synonym Full Names:	heat shock transcription factor 1
NCBI Official Symbol:	HSF1
NCBI Official Synonym Symbols:	HSTF1
NCBI Protein Information:	heat shock factor protein 1; HSF 1; HSTF 1
UniProt Protein Name:	Heat shock factor protein 1
UniProt Synonym Protein Names:	Heat shock transcription factor 1; HSTF 1
Protein Family:	Heat shock factor protein
UniProt Gene Name:	HSF1
UniProt Entry Name:	HSF1_HUMAN

Component	Quantity
96-Well Cell Culture Clear-Bottom Microplate	2 plates
10X TBS	24 mL
Quenching Buffer	24 mL
Blocking Buffer	50 mL
15X Wash Buffer	50 mL
Primary Antibody Diluent	12 mL
100x Anti-Phospho Target Antibody	60 µL
100x Anti-Target Antibody	60 µL
Anti-GAPDH Antibody	60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody	12 mL
HRP-Conjugated Anti-Mouse IgG Antibody	12 mL
SDS Solution	12 mL
Stop Solution	24 mL
Ready-to-Use Substrate	12 mL
Crystal Violet Solution	12 mL
Adhesive Plate Seals	2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
Graph paper or computer software capable of generating or displaying logarithmic functions
Absorbent papers or vacuum aspirator
Test tubes or microfuge tubes capable of storing ≥1 ml
Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
Orbital shaker (optional)
Deionized or sterile water

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

Step	Procedure
1.	Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37 °C prior to adding cells.
2.	Incubate the cells for overnight at 37 °C, 5% CO2.
3.	Treat the cells as desired.
4.	Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.	Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.	Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4 °C for a week.
7.	Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.	Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.	Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.	Add 50 µL of 1x primary antibodies Anti-HSF1 (Phospho-Ser121) Antibody, Anti-HSF1 Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4 °C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.	Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.	Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.	Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)