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PDLIM1 Colorimetric Cell-Based ELISA

SKU:
CBCAB00919
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
Frequently bought together:

Description

system_update_altDatasheetMSDS

PDLIM1 Colorimetric Cell-Based ELISA

The PDLIM1 Colorimetric Cell-Based ELISA Kit is specifically designed for the detection of PDLIM1 levels in cell lysates. This kit offers high sensitivity and accuracy, providing researchers with reliable and reproducible results for a variety of applications.PDLIM1, also known as CLP36, is a PDZ and LIM domain protein that plays a crucial role in cell signaling and actin cytoskeleton organization. Dysregulation of PDLIM1 has been implicated in various diseases, including cancer and cardiovascular disorders, making it a valuable biomarker for studying these conditions and developing therapeutic interventions.

With easy-to-follow protocols and quick assay procedures, the PDLIM1 Colorimetric Cell-Based ELISA Kit is an essential tool for researchers interested in studying PDLIM1 function and its role in disease pathways. Upgrade your research capabilities with this high-quality kit from AssayGenie.

Product Name:PDLIM1 Colorimetric Cell-Based ELISA
Product Code:CBCAB00919
ELISA Type:Cell-Based
Target:PDLIM1
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The PDLIM1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect PDLIM1 protein expression profile in cells. The kit can be used for measuring the relative amounts of PDLIM1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on PDLIM1.

Qualitative determination of PDLIM1 concentration is achieved by an indirect ELISA format. In essence, PDLIM1 is captured by PDLIM1-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: 9124, UniProt ID: O00151, OMIM: 605900, Unigene: Hs.368525
Gene Symbol:PDLIM1
Sub Type:None
UniProt Protein Function:CLIM1: a cytoskeletal adaptor protein that bring other proteins to the cytoskeleton. Interacts with alpha-actinins 1, 2 and 4. Recruits the Clik1 kinase to actin stress fibers in nonmuscle cells. Strongly expressed in the heart and skeletal muscle, moderately expressed in the spleen, small intestine, colon, placenta, and lung. A lower level expression is seen in liver, thymus, kidney, prostate and pancreas and is not found in the brain, testis, ovary, and peripheral blood leukocytes.
UniProt Protein Details:

Protein type:Cytoskeletal

Chromosomal Location of Human Ortholog: 10q23.1

Cellular Component: transcription factor complex; cytoskeleton; focal adhesion; cytoplasm

Molecular Function:zinc ion binding; transcription coactivator activity

Biological Process: regulation of transcription, DNA-dependent; response to hypoxia; response to oxidative stress

NCBI Summary:This gene encodes a member of the enigma protein family. The protein contains two protein interacting domains, a PDZ domain at the amino terminal end and one to three LIM domains at the carboxyl terminal. It is a cytoplasmic protein associated with the cytoskeleton. The protein may function as an adapter to bring other LIM-interacting proteins to the cytoskeleton. Pseudogenes associated with this gene are located on chromosomes 3, 14 and 17. [provided by RefSeq, Oct 2012]
UniProt Code:O00151
NCBI GenInfo Identifier:20178312
NCBI Gene ID:9124
NCBI Accession:O00151.4
UniProt Secondary Accession:O00151,Q5VZH5, Q9BPZ9, B2RBS6,
UniProt Related Accession:O00151
Molecular Weight:329
NCBI Full Name:PDZ and LIM domain protein 1
NCBI Synonym Full Names:PDZ and LIM domain 1
NCBI Official Symbol:PDLIM1  
NCBI Official Synonym Symbols:CLIM1; CLP36; CLP-36; hCLIM1; HEL-S-112  
NCBI Protein Information:PDZ and LIM domain protein 1; elfin; LIM domain protein CLP-36; epididymis secretory protein Li 112; carboxyl terminal LIM domain protein 1
UniProt Protein Name:PDZ and LIM domain protein 1
UniProt Synonym Protein Names:C-terminal LIM domain protein 1; Elfin; LIM domain protein CLP-36
Protein Family:PDZ and LIM domain protein
UniProt Gene Name:PDLIM1  
UniProt Entry Name:PDLI1_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-PDLIM1 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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