Histone H1 Colorimetric Cell-Based ELISA Kit (CBCAB00312)

Product Name:	Histone H1 Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00312
ELISA Type:	Cell-Based
Target:	Histone H1
Reactivity:	Human, Mouse
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The Histone H1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Histone H1 protein expression profile in cells. The kit can be used for measuring the relative amounts of Histone H1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Histone H1.

Qualitative determination of Histone H1 concentration is achieved by an indirect ELISA format. In essence, Histone H1 is captured by Histone H1-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: 3005/3024/3006/3007/3008/3009, UniProt ID: P16401/P16402/P10412, OMIM: 142708/142709/142710/142711/142210/142220, Unigene: Hs.740570/Hs.150206/Hs.7644/Hs.131956/Hs.136857/Hs.248133
Gene Symbol:	HIST1H1B
Sub Type:	None

UniProt Protein Function:	H1B: Histone H1 protein binds to linker DNA between nucleosomes forming the macromolecular structure known as the chromatin fiber. Histones H1 are necessary for the condensation of nucleosome chains into higher-order structured fibers. Acts also as a regulator of individual gene transcription through chromatin remodeling, nucleosome spacing and DNA methylation. Belongs to the histone H1/H5 family.
UniProt Protein Details:	Protein type:DNA-binding Chromosomal Location of Human Ortholog: 6p22.1 Cellular Component: nuclear chromatin; nuclear heterochromatin; nucleosome Molecular Function:chromatin DNA binding; histone deacetylase binding Biological Process: establishment and/or maintenance of chromatin architecture; negative regulation of transcription from RNA polymerase II promoter; nucleosome assembly; positive regulation of cell growth; positive regulation of histone H3-K9 methylation; protein stabilization
NCBI Summary:	Histones are basic nuclear proteins responsible for nucleosome structure of the chromosomal fiber in eukaryotes. Two molecules of each of the four core histones (H2A, H2B, H3, and H4) form an octamer, around which approximately 146 bp of DNA is wrapped in repeating units, called nucleosomes. The linker histone, H1, interacts with linker DNA between nucleosomes and functions in the compaction of chromatin into higher order structures. This gene is intronless and encodes a replication-dependent histone that is a member of the histone H1 family. Transcripts from this gene lack polyA tails but instead contain a palindromic termination element. This gene is found in the small histone gene cluster on chromosome 6p22-p21.3. [provided by RefSeq, Aug 2015]
UniProt Code:	P16401
NCBI GenInfo Identifier:	19856407
NCBI Gene ID:	3009
NCBI Accession:	P16401.3
UniProt Secondary Accession:	P16401,Q14529, Q3MJ42,
UniProt Related Accession:	P16401
Molecular Weight:	22,580 Da
NCBI Full Name:	Histone H1.5
NCBI Synonym Full Names:	histone cluster 1, H1b
NCBI Official Symbol:	HIST1H1B
NCBI Official Synonym Symbols:	H1; H1B; H1.5; H1F5; H1s-3
NCBI Protein Information:	histone H1.5
UniProt Protein Name:	Histone H1.5
UniProt Synonym Protein Names:	Histone H1a; Histone H1b; Histone H1s-3
Protein Family:	Histone
UniProt Gene Name:	HIST1H1B
UniProt Entry Name:	H15_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-Histone H1 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.)