ADCK4 Colorimetric Cell-Based ELISA

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SKU:
CBCAB01177
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
Frequently bought together:

Description

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ADCK4 Colorimetric Cell-Based ELISA

The ADCY1 Human Colorimetric Cell-Based ELISA Kit is a cutting-edge solution for measuring intracellular levels of ADCY1 in cell cultures. With its high sensitivity and specificity, this kit provides accurate and reliable results for researchers studying the role of ADCY1 in various cellular processes.ADCY1, also known as adenylate cyclase 1, plays a crucial role in signal transduction pathways by converting ATP into cyclic AMP. Dysregulation of ADCY1 has been linked to a variety of diseases, including metabolic disorders and cancer, making it a valuable target for drug development and therapeutic interventions.

The ADCY1 Human Colorimetric Cell-Based ELISA Kit is a must-have tool for researchers looking to gain deeper insights into the function of ADCY1 in cellular signaling and disease pathogenesis. Order yours today and unlock the potential of ADCY1 research in your laboratory.

Product Name:ADCK4 Colorimetric Cell-Based ELISA
Product Code:CBCAB01177
ELISA Type:Cell-Based
Target:ADCK4
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The ADCK4 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect ADCK4 protein expression profile in cells. The kit can be used for measuring the relative amounts of ADCK4 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on ADCK4.

Qualitative determination of ADCK4 concentration is achieved by an indirect ELISA format. In essence, ADCK4 is captured by ADCK4-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: 79934, UniProt ID: Q96D53, OMIM: 0, Unigene: Hs.130712
Gene Symbol:ADCK4
Sub Type:None
UniProt Protein Function:ADCK4: The function of this protein is not yet clear. It is not known if it has protein kinase activity and what type of substrate it would phosphorylate (Ser, Thr or Tyr). Belongs to the protein kinase superfamily. ADCK protein kinase family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:ABC1 family; ABC1-A subfamily; ATYPICAL group; EC 2.7.-.-; EC 2.7.11.-; Kinase, protein; Membrane protein, integral; Protein kinase, Ser/Thr (non-receptor); Protein kinase, atypical

Chromosomal Location of Human Ortholog: 19q13.2

Cellular Component: cytosol; integral component of membrane; mitochondrial membrane; mitochondrion; plasma membrane

Molecular Function:ATP binding; protein kinase activity

Biological Process: cerebellar Purkinje cell layer morphogenesis; protein amino acid phosphorylation; ubiquinone biosynthetic process

Disease: Nephrotic Syndrome, Type 9

NCBI Summary:This gene encodes a protein with two copies of a domain found in protein kinases. The encoded protein has a complete protein kinase catalytic domain, and a truncated domain that contains only the active and binding sites of the protein kinase domain, however, it is not known whether the protein has any kinase activity. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Sep 2011]
UniProt Code:Q96D53
NCBI GenInfo Identifier:217416386
NCBI Gene ID:79934
NCBI Accession:NP_001136027.1
UniProt Secondary Accession:Q96D53,Q8TAJ1, Q9HA52,
UniProt Related Accession:Q96D53
Molecular Weight:60 kDa
NCBI Full Name:atypical kinase COQ8B, mitochondrial isoform b
NCBI Synonym Full Names:coenzyme Q8B
NCBI Official Symbol:COQ8B  
NCBI Official Synonym Symbols:ADCK4; NPHS9  
NCBI Protein Information:atypical kinase COQ8B, mitochondrial
UniProt Protein Name:Atypical kinase COQ8B, mitochondrial
UniProt Synonym Protein Names:AarF domain-containing protein kinase 4
UniProt Gene Name:COQ8B  
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-ADCK4 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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