null

PFDN1 Colorimetric Cell-Based ELISA

SKU:
CBCAB00946
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
€599
Frequently bought together:

Description

system_update_altDatasheetMSDS

PFDN1 Colorimetric Cell-Based ELISA

The PFDN1 Colorimetric Cell-Based ELISA is a cutting-edge assay kit designed for the accurate quantification of PFDN1 levels in cell lysates. This kit offers high sensitivity and specificity, allowing for precise and reliable results in a wide range of research applications.PFDN1, also known as prefoldin subunit 1, is a key protein involved in protein folding and chaperone activity within cells. Dysregulation of PFDN1 has been implicated in various diseases, including cancer, neurodegenerative disorders, and autoimmune diseases, underscoring its importance as a potential biomarker for studying and developing treatments for these conditions.

The PFDN1 Colorimetric Cell-Based ELISA kit is easy to use and provides quick results, making it ideal for researchers looking to accurately measure PFDN1 levels in cell samples.Upgrade your research with the PFDN1 Colorimetric Cell-Based ELISA kit and unlock new insights into cellular processes and disease mechanisms.

Product Name:PFDN1 Colorimetric Cell-Based ELISA
Product Code:CBCAB00946
ELISA Type:Cell-Based
Target:PFDN1
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The PFDN1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect PFDN1 protein expression profile in cells. The kit can be used for measuring the relative amounts of PFDN1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on PFDN1.

Qualitative determination of PFDN1 concentration is achieved by an indirect ELISA format. In essence, PFDN1 is captured by PFDN1-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: 5201, UniProt ID: O60925, OMIM: 604897, Unigene: Hs.483564
Gene Symbol:PFDN1
Sub Type:None
UniProt Protein Function:PFDN1: Binds specifically to cytosolic chaperonin (c-CPN) and transfers target proteins to it. Binds to nascent polypeptide chain and promotes folding in an environment in which there are many competing pathways for nonnative proteins. Belongs to the prefoldin subunit beta family.
UniProt Protein Details:

Protein type:Chaperone

Chromosomal Location of Human Ortholog: 5q31

Molecular Function:transcription factor activity

Biological Process: cell cycle

NCBI Summary:This gene encodes a member of the prefoldin beta subunit family. The encoded protein is one of six subunits of prefoldin, a molecular chaperone complex that binds and stabilizes newly synthesized polypeptides, thereby allowing them to fold correctly. The complex, consisting of two alpha and four beta subunits, forms a double beta barrel assembly with six protruding coiled-coils. [provided by RefSeq, Jul 2008]
UniProt Code:O60925
NCBI GenInfo Identifier:22261811
NCBI Gene ID:5201
NCBI Accession:O60925.2
UniProt Secondary Accession:O60925,Q53F95, Q96EX6, B2RD02,
UniProt Related Accession:O60925
Molecular Weight:14,210 Da
NCBI Full Name:Prefoldin subunit 1
NCBI Synonym Full Names:prefoldin subunit 1
NCBI Official Symbol:PFDN1  
NCBI Official Synonym Symbols:PDF; PFD1  
NCBI Protein Information:prefoldin subunit 1
UniProt Protein Name:Prefoldin subunit 1
Protein Family:Prefoldin
UniProt Gene Name:PFDN1  
UniProt Entry Name:PFD1_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-PFDN1 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