KLF11 Colorimetric Cell-Based ELISA (CBCAB00927)

Product Name:	KLF11 Colorimetric Cell-Based ELISA
Product Code:	CBCAB00927
ELISA Type:	Cell-Based
Target:	KLF11
Reactivity:	Human, Mouse
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The KLF11 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect KLF11 protein expression profile in cells. The kit can be used for measuring the relative amounts of KLF11 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on KLF11.

Qualitative determination of KLF11 concentration is achieved by an indirect ELISA format. In essence, KLF11 is captured by KLF11-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary 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: 8462, UniProt ID: O14901, OMIM: 603301/606391/610508, Unigene: Hs.12229/Hs.694968
Gene Symbol:	KLF11
Sub Type:	None

UniProt Protein Function:	TIEG2: Transcription factor. Activates the epsilon- and gamma- globin gene promoters and, to a much lower degree, the beta-globin gene and represses promoters containing SP1-like binding inhibiting cell growth. Represses transcription of SMAD7 which enhances TGF-beta signaling. Induces apoptosis. Defects in KLF11 are the cause of maturity-onset diabetes of the young type 7 (MODY7). MODY is a form of diabetes that is characterized by an autosomal dominant mode of inheritance, onset in childhood or early adulthood (usually before 25 years of age), a primary defect in insulin secretion and frequent insulin-independence at the beginning of the disease. Belongs to the Sp1 C2H2-type zinc-finger protein family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:DNA-binding; C2H2-type zinc finger protein; Transcription factor Chromosomal Location of Human Ortholog: 2p25 Molecular Function:protein binding; transcription factor activity Biological Process: G1/S-specific transcription in mitotic cell cycle; negative regulation of cell proliferation; negative regulation of transcription from RNA polymerase II promoter; positive regulation of apoptosis; transcription from RNA polymerase II promoter Disease: Maturity-onset Diabetes Of The Young, Type 7
NCBI Summary:	The protein encoded by this gene is a zinc finger transcription factor that binds to SP1-like sequences in epsilon- and gamma-globin gene promoters. This binding inhibits cell growth and causes apoptosis. Defects in this gene are a cause of maturity-onset diabetes of the young type 7 (MODY7). Three transcript variants encoding two different isoforms have been found for this gene. [provided by RefSeq, Apr 2010]
UniProt Code:	O14901
NCBI GenInfo Identifier:	11387048
NCBI Gene ID:	8462
NCBI Accession:	O14901.2
UniProt Secondary Accession:	O14901,Q9EPF4, B4DZE7,
UniProt Related Accession:	O14901
Molecular Weight:	53,329 Da
NCBI Full Name:	Krueppel-like factor 11
NCBI Synonym Full Names:	Kruppel like factor 11
NCBI Official Symbol:	KLF11
NCBI Official Synonym Symbols:	FKLF; FKLF1; MODY7; TIEG2; Tieg3
NCBI Protein Information:	Krueppel-like factor 11
UniProt Protein Name:	Krueppel-like factor 11
UniProt Synonym Protein Names:	Transforming growth factor-beta-inducible early growth response protein 2; TGFB-inducible early growth response protein 2; TIEG-2
Protein Family:	Krueppel-like factor
UniProt Gene Name:	KLF11
UniProt Entry Name:	KLF11_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-KLF11 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.)