null

HSP40 Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00701
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Metabolism
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
Frequently bought together:

Description

system_update_altDatasheetMSDS

HSP40 Colorimetric Cell-Based ELISA Kit

The HSP40 Colorimetric Cell-Based ELISA Kit offered by Assay Genie is a powerful tool for researchers looking to accurately measure the levels of HSP40 (Heat Shock Protein 40) in cell culture supernatants. With its high sensitivity and specificity, this kit provides reliable and reproducible results, making it ideal for a wide range of applications.HSP40 is a chaperone protein that plays a crucial role in protein folding and cellular stress response. Dysregulation of HSP40 has been linked to various diseases, including cancer, neurodegenerative disorders, and cardiovascular diseases.

By measuring HSP40 levels, researchers can gain valuable insights into cellular stress and disease pathways, potentially identifying new therapeutic targets.With the HSP40 Colorimetric Cell-Based ELISA Kit, researchers can confidently study the role of HSP40 in health and disease, paving the way for new discoveries and therapeutic interventions. Order yours today from Assay Genie and take your research to the next level.

Product Name:HSP40 Colorimetric Cell-Based ELISA
Product Code:CBCAB00701
ELISA Type:Cell-Based
Target:HSP40
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The HSP40 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect HSP40 protein expression profile in cells. The kit can be used for measuring the relative amounts of HSP40 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on HSP40.

Qualitative determination of HSP40 concentration is achieved by an indirect ELISA format. In essence, HSP40 is captured by HSP40-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: 3337, UniProt ID: P25685, OMIM: 604572, Unigene: Hs.515210
Gene Symbol:DNAJB1
Sub Type:None
UniProt Protein Function:Interacts with HSP70 and can stimulate its ATPase activity. Stimulates the association between HSC70 and HIP.
NCBI Summary:This gene encodes a member of the DnaJ or Hsp40 (heat shock protein 40 kD) family of proteins. DNAJ family members are characterized by a highly conserved amino acid stretch called the 'J-domain' and function as one of the two major classes of molecular chaperones involved in a wide range of cellular events, such as protein folding and oligomeric protein complex assembly. The encoded protein is a molecular chaperone that stimulates the ATPase activity of Hsp70 heat-shock proteins in order to promote protein folding and prevent misfolded protein aggregation. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Sep 2015]
UniProt Code:P25685
NCBI GenInfo Identifier:1706473
NCBI Gene ID:3337
NCBI Accession:P25685.4
UniProt Secondary Accession:P25685,B4DX52,
UniProt Related Accession:P25685
Molecular Weight:340
NCBI Full Name:DnaJ homolog subfamily B member 1
NCBI Synonym Full Names:DnaJ (Hsp40) homolog, subfamily B, member 1
NCBI Official Symbol:DNAJB1  
NCBI Official Synonym Symbols:Hdj1; Sis1; HSPF1; Hsp40; RSPH16B  
NCBI Protein Information:dnaJ homolog subfamily B member 1; human DnaJ protein 1; dnaJ protein homolog 1; radial spoke 16 homolog B; heat shock 40 kDa protein 1
UniProt Protein Name:DnaJ homolog subfamily B member 1
UniProt Synonym Protein Names:DnaJ protein homolog 1; Heat shock 40 kDa protein 1; HSP40; Heat shock protein 40; Human DnaJ protein 1; hDj-1
Protein Family:DnaJ homolog subfamily
UniProt Gene Name:DNAJB1  
UniProt Entry Name:DNJB1_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-HSP40 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.)

Related Products