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Pax-5 Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00802
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Developmental Biology
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
€599
Frequently bought together:

Description

system_update_altDatasheetMSDS

Pax-5 Colorimetric Cell-Based ELISA Kit

The PAX-5 Colorimetric Cell-Based ELISA Kit is a comprehensive tool for the precise measurement of PAX-5 levels in cell lysates and tissue samples. This kit offers exceptional sensitivity and accuracy, guaranteeing consistent and dependable results that are well-suited for various research purposes.PAX-5 is a key transcription factor that plays a critical role in B-cell development and function. Dysregulation of PAX-5 has been linked to various hematological malignancies, making it a valuable target for studying the pathogenesis of these diseases and identifying potential therapeutic strategies.

With its advanced technology and user-friendly design, the PAX-5 Colorimetric Cell-Based ELISA Kit is an indispensable resource for researchers looking to explore the intricate mechanisms underlying B-cell biology and develop novel therapies for B-cell-related disorders.

Product Name:Pax-5 Colorimetric Cell-Based ELISA
Product Code:CBCAB00802
ELISA Type:Cell-Based
Target:Pax-5
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The Pax-5 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Pax-5 protein expression profile in cells. The kit can be used for measuring the relative amounts of Pax-5 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Pax-5.

Qualitative determination of Pax-5 concentration is achieved by an indirect ELISA format. In essence, Pax-5 is captured by Pax-5-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: 5079, UniProt ID: Q02548, OMIM: 167414, Unigene: Hs.654464
Gene Symbol:PAX5
Sub Type:None
UniProt Protein Function:May play an important role in B-cell differentiation as well as neural development and spermatogenesis. Involved in the regulation of the CD19 gene, a B-lymphoid-specific target gene.
NCBI Summary:This gene encodes a member of the paired box (PAX) family of transcription factors. The central feature of this gene family is a novel, highly conserved DNA-binding motif, known as the paired box. Paired box transcription factors are important regulators in early development, and alterations in the expression of their genes are thought to contribute to neoplastic transformation. This gene encodes the B-cell lineage specific activator protein that is expressed at early, but not late stages of B-cell differentiation. Its expression has also been detected in developing CNS and testis and so the encoded protein may also play a role in neural development and spermatogenesis. This gene is located at 9p13, which is involved in t(9;14)(p13;q32) translocations recurring in small lymphocytic lymphomas of the plasmacytoid subtype, and in derived large-cell lymphomas. This translocation brings the potent E-mu enhancer of the IgH gene into close proximity of the PAX5 promoter, suggesting that the deregulation of transcription of this gene contributes to the pathogenesis of these lymphomas. Alternative splicing results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Jul 2013]
UniProt Code:Q02548
NCBI GenInfo Identifier:417449
NCBI Gene ID:5079
NCBI Accession:Q02548.1
UniProt Secondary Accession:Q02548,O75933, Q5SFM2, Q6S728, A3QVP6, A3QVP7, A3QVP8 C0KTF6, C0KTF7, C0KTF8, C0KTF9, C0KTG0,
UniProt Related Accession:Q02548
Molecular Weight:34,132 Da
NCBI Full Name:Paired box protein Pax-5
NCBI Synonym Full Names:paired box 5
NCBI Official Symbol:PAX5  
NCBI Official Synonym Symbols:ALL3; BSAP  
NCBI Protein Information:paired box protein Pax-5
UniProt Protein Name:Paired box protein Pax-5
UniProt Synonym Protein Names:B-cell-specific transcription factor; BSAP
Protein Family:Paired box protein
UniProt Gene Name:PAX5  
UniProt Entry Name:PAX5_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-Pax-5 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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