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APC (Phospho-Ser2054) Colorimetric Cell-Based ELISA Kit (CBCAB01385)

SKU:
CBCAB01385
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Metabolism
Reactivity:
Human
Detection Method:
Colorimetric
$1,152

Description

system_update_altDatasheetMSDS

APC (Phospho-Ser2054)Colorimetric Cell-Based ELISA Kit

The APC (Phospho-Ser2054) Colorimetric Cell-Based ELISA Kit is a cutting-edge tool for studying the phosphorylation of APC at Ser2054 in cell-based experiments. This kit offers high sensitivity and accuracy, allowing researchers to track the activation of the APC protein with precision.APC, or adenomatous polyposis coli, is a key player in cell cycle regulation and tumor suppression. Phosphorylation of APC at Ser2054 is known to modulate its function and may play a role in various diseases, including cancer. Understanding this phosphorylation event is critical for unraveling the complex mechanisms of cell growth and division.

With the APC (Phospho-Ser2054) Colorimetric Cell-Based ELISA Kit, researchers can delve into the intricate world of cell signaling pathways and explore the potential therapeutic implications of targeting APC phosphorylation. This kit is a valuable asset for studies in cancer biology, cell biology, and drug discovery, offering reliable and reproducible results for a wide range of applications.

Product Name:APC (Phospho-Ser2054) Colorimetric Cell-Based ELISA
Product Code:CBCAB01385
ELISA Type:Cell-Based
Target:APC (Phospho-Ser2054)
Reactivity:Human
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The APC (Phospho-Ser2054) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect APC protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated APC in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on APC phosphorylation.

Qualitative determination of APC (Phospho-Ser2054) concentration is achieved by an indirect ELISA format. In essence, APC (Phospho-Ser2054) is captured by APC (Phospho-Ser2054)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø 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: 324, UniProt ID: P25054, OMIM: 135290/155255/175100/276300/611731, Unigene: Hs.158932
Gene Symbol:APC
Sub Type:Phospho
UniProt Code:P25054
NCBI GenInfo Identifier:97535708
NCBI Gene ID:324
NCBI Accession:P25054.2
UniProt Secondary Accession:P25054,Q15162, Q15163, Q93042, D3DT03,
UniProt Related Accession:P25054
Molecular Weight:461
NCBI Full Name:Adenomatous polyposis coli protein
NCBI Synonym Full Names:adenomatous polyposis coli
NCBI Official Symbol:APC  
NCBI Official Synonym Symbols:GS; DP2; DP3; BTPS2; DP2.5; PPP1R46  
NCBI Protein Information:adenomatous polyposis coli protein; deleted in polyposis 2.5; adenomatosis polyposis coli tumor suppressor; protein phosphatase 1, regulatory subunit 46
UniProt Protein Name:Adenomatous polyposis coli protein
UniProt Synonym Protein Names:Deleted in polyposis 2.5
Protein Family:APC-related protein
UniProt Gene Name:APC  
UniProt Entry Name:APC_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-APC (Phospho-Ser2054) Antibody, Anti-APC 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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