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HP1gamma Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00697
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Cell Biology
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheetMSDS

HP1gamma Colorimetric Cell-Based ELISA Kit

The HP1gamma Colorimetric Cell-Based ELISA Kit is a powerful tool designed for the detection of HP1gamma levels in cell lysates and tissue extracts. This kit offers high sensitivity and specificity, providing accurate and reliable results for a variety of research applications.HP1gamma is a key protein involved in chromatin regulation and gene expression, playing a critical role in cellular processes such as cell division, DNA repair, and development. Dysregulation of HP1gamma has been associated with various diseases, including cancer, neurodegenerative disorders, and metabolic diseases.

Therefore, studying HP1gamma levels can provide valuable insights into disease mechanisms and potential therapeutic targets.With its easy-to-use protocol and robust performance, the HP1gamma Colorimetric Cell-Based ELISA Kit is a valuable tool for researchers investigating the role of HP1gamma in health and disease. Get accurate and reproducible results with this reliable ELISA kit from Assaygenie.

Product Name:HP1gamma Colorimetric Cell-Based ELISA
Product Code:CBCAB00697
ELISA Type:Cell-Based
Target:HP1gamma
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The HP1gamma Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect HP1gamma protein expression profile in cells. The kit can be used for measuring the relative amounts of HP1gamma in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on HP1gamma.

Qualitative determination of HP1gamma concentration is achieved by an indirect ELISA format. In essence, HP1gamma is captured by HP1gamma-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: 11335, UniProt ID: Q13185, OMIM: 604477, Unigene: Hs.381189
Gene Symbol:CBX3
Sub Type:None
UniProt Protein Function:CBX3: Seems to be involved in transcriptional silencing in heterochromatin-like complexes. Recognizes and binds histone H3 tails methylated at 'Lys-9', leading to epigenetic repression. May contribute to the association of the heterochromatin with the inner nuclear membrane through its interaction with lamin B receptor (LBR). Involved in the formation of functional kinetochore through interaction with MIS12 complex proteins. Binds directly to CHAF1A. Interacts with histone H3 methylated at 'Lys-9'. Part of the E2F6.com-1 complex in G0 phase composed of E2F6, MGA, MAX, TFDP1, CBX3, BAT8, EUHMTASE1, RING1, RNF2, MBLR, L3MBTL2 and YAF2. Interacts with LBR, INCENP, TRIM28/TIF1B, SUV420H1, SUV420H2 and SP100. Interacts with TIF1A. Interacts with MIS12 and DSN1. Can interact directly with CBX5 via the chromoshadow domain. Interacts with POGZ. Interacts with CHAMP1. Interacts with ASXL1.
UniProt Protein Details:

Protein type:DNA-binding

Chromosomal Location of Human Ortholog: 7p15.2

Cellular Component: chromatin; chromocenter; chromosome, pericentric region; nuclear chromosome, telomeric region; nuclear envelope; nuclear heterochromatin; nuclear inner membrane; nucleus; spindle

Molecular Function:enzyme binding; identical protein binding; protein binding; protein domain specific binding

Biological Process: chromatin remodeling; negative regulation of transcription, DNA-dependent; rhythmic process; transcription, DNA-dependent

NCBI Summary:At the nuclear envelope, the nuclear lamina and heterochromatin are adjacent to the inner nuclear membrane. The protein encoded by this gene binds DNA and is a component of heterochromatin. This protein also can bind lamin B receptor, an integral membrane protein found in the inner nuclear membrane. The dual binding functions of the encoded protein may explain the association of heterochromatin with the inner nuclear membrane. This protein binds histone H3 tails methylated at Lys-9 sites. This protein is also recruited to sites of ultraviolet-induced DNA damage and double-strand breaks. Two transcript variants encoding the same protein but differing in the 5' UTR, have been found for this gene.[provided by RefSeq, Mar 2011]
UniProt Code:Q13185
NCBI GenInfo Identifier:116241284
NCBI Gene ID:11335
NCBI Accession:Q13185.4
UniProt Secondary Accession:Q13185,Q96CD7, Q99409, Q9BVS3, Q9P0Z6,
UniProt Related Accession:Q13185
Molecular Weight:20,811 Da
NCBI Full Name:Chromobox protein homolog 3
NCBI Synonym Full Names:chromobox 3
NCBI Official Symbol:CBX3  
NCBI Official Synonym Symbols:HECH; HP1-GAMMA; HP1Hs-gamma  
NCBI Protein Information:chromobox protein homolog 3
UniProt Protein Name:Chromobox protein homolog 3
UniProt Synonym Protein Names:HECH; Heterochromatin protein 1 homolog gamma; HP1 gamma; Modifier 2 protein
Protein Family:Chromobox protein
UniProt Gene Name:CBX3  
UniProt Entry Name:CBX3_HUMAN
ComponentQuantity
96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 mL
Quenching Buffer24 mL
Blocking Buffer50 mL
15X Wash Buffer50 mL
Primary Antibody Diluent12 mL
100x Anti-Phospho Target Antibody 60 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 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.

StepProcedure
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-HP1gamma 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.)

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