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HSP10 Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00313
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Metabolism
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheetMSDS

HSP10 Colorimetric Cell-Based ELISA Kit

The HSP10 Colorimetric Cell-based ELISA Kit is a valuable tool for studying Heat Shock Protein 10 levels in cell samples. This kit offers high sensitivity and specificity, allowing for accurate and reproducible results in a variety of research applications.Heat Shock Protein 10 (HSP10) is an essential chaperone protein involved in cellular stress response. It plays a critical role in protein folding, stabilization, and transport, making it a key player in maintaining cellular homeostasis. Abnormal HSP10 levels have been linked to various diseases such as cancer, neurodegenerative disorders, and autoimmune conditions, highlighting its importance as a potential biomarker and therapeutic target.

With the HSP10 Colorimetric Cell-based ELISA Kit, researchers can easily quantify HSP10 levels in cell samples, providing valuable insights into cellular stress response mechanisms and potential disease pathways. This kit is user-friendly, efficient, and reliable, making it an indispensable tool for any lab studying HSP10 and its role in human health and disease.

Product Name:HSP10 Colorimetric Cell-Based ELISA
Product Code:CBCAB00313
ELISA Type:Cell-Based
Target:HSP10
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The HSP10 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect HSP10 protein expression profile in cells. The kit can be used for measuring the relative amounts of HSP10 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on HSP10.

Qualitative determination of HSP10 concentration is achieved by an indirect ELISA format. In essence, HSP10 is captured by HSP10-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: 3336, UniProt ID: P61604, OMIM: 600141, Unigene: Hs.1197
Gene Symbol:HSPE1
Sub Type:None
UniProt Protein Function:HSPE1: Eukaryotic CPN10 homolog which is essential for mitochondrial protein biogenesis, together with CPN60. Binds to CPN60 in the presence of Mg-ATP and suppresses the ATPase activity of the latter. Belongs to the GroES chaperonin family.
UniProt Protein Details:

Protein type:Mitochondrial

Chromosomal Location of Human Ortholog: 2q33.1

Cellular Component: membrane; mitochondrial matrix; mitochondrion

Molecular Function:ATP binding; chaperone binding; protein binding; unfolded protein binding

Biological Process: caspase activation; osteoblast differentiation; protein folding; response to unfolded protein

NCBI Summary:This gene encodes a major heat shock protein which functions as a chaperonin. Its structure consists of a heptameric ring which binds to another heat shock protein in order to form a symmetric, functional heterodimer which enhances protein folding in an ATP-dependent manner. This gene and its co-chaperonin, HSPD1, are arranged in a head-to-head orientation on chromosome 2. Naturally occurring read-through transcription occurs between this locus and the neighboring locus MOBKL3.[provided by RefSeq, Feb 2011]
UniProt Code:P61604
NCBI GenInfo Identifier:47606335
NCBI Gene ID:3336
NCBI Accession:P61604.2
UniProt Secondary Accession:P61604,O95421, Q04984, Q53X54, Q9UDH0,
UniProt Related Accession:P61604
Molecular Weight:10,932 Da
NCBI Full Name:10 kDa heat shock protein, mitochondrial
NCBI Synonym Full Names:heat shock protein family E (Hsp10) member 1
NCBI Official Symbol:HSPE1  
NCBI Official Synonym Symbols:EPF; CPN10; GROES; HSP10  
NCBI Protein Information:10 kDa heat shock protein, mitochondrial
UniProt Protein Name:10 kDa heat shock protein, mitochondrial
UniProt Synonym Protein Names:10 kDa chaperonin; Chaperonin 10; CPN10; Early-pregnancy factor; EPF
UniProt Gene Name:HSPE1  
UniProt Entry Name:CH10_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-HSP10 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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