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LIMK1/2 (Phospho-Thr505) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB01561
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

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LIMK1/2 (Phospho-Thr505)Colorimetric Cell-Based ELISA Kit

The LIMK1 (LIM domain kinase 1) Phospho Thr505 Colorimetric Cell-based ELISA Kit is a reliable tool for the quantitative detection of LIMK1 phosphorylated at threonine 505 in cell lysates. This kit offers high sensitivity and specificity, allowing for accurate and reproducible results in a variety of research applications.LIMK1 is a key regulator of actin dynamics and cell motility, playing a crucial role in processes such as cell migration, adhesion, and invasion. Phosphorylation of LIMK1 at threonine 505 is known to affect its activity and function, making it an important target for studying cellular signaling pathways and potential therapeutic interventions.

By utilizing the LIMK1 Phospho Thr505 Colorimetric Cell-based ELISA Kit, researchers can gain valuable insights into the role of LIMK1 phosphorylation in various cellular processes and diseases, offering a deeper understanding of its potential implications in cancer, neurological disorders, and other conditions.

Product Name:LIMK1/2 (Phospho-Thr505) Colorimetric Cell-Based ELISA
Product Code:CBCAB01561
ELISA Type:Cell-Based
Target:LIMK1/2 (Phospho-Thr505)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The LIMK1/2 (Phospho-Thr505) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect LIMK1/2 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated LIMK1/2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on LIMK1/2 phosphorylation.

Qualitative determination of LIMK1/2 (Phospho-Thr505) concentration is achieved by an indirect ELISA format. In essence, LIMK1/2 (Phospho-Thr505) is captured by LIMK1/2 (Phospho-Thr505)-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: 3985, UniProt ID: P53671, OMIM: 601988, Unigene: Hs.474596
Gene Symbol:LIMK2
Sub Type:Phospho
UniProt Protein Function:LIMK2: a TKL kinase of the LISK family that regulates actin dynamics. Contains two N-terminal LIM motifs. Phosphorylated and activated by PAK1 and ROCK, downstream effectors of Rho. Phosphorylates cofilin, inhibiting its actin-depolymerizing activity. May contributes to Rho-induced reorganization of the actin cytoskeleton. Two alternative splice variants have been identified.
UniProt Protein Details:

Protein type:Protein kinase, TKL; Protein kinase, Ser/Thr (non-receptor); EC 2.7.11.1; Kinase, protein; TKL group; LISK family; LIMK subfamily

Chromosomal Location of Human Ortholog: 22q12.2

Cellular Component: cis-Golgi network; cytoplasm; nucleus

Molecular Function:protein serine/threonine kinase activity; protein binding; zinc ion binding; protein heterodimerization activity; ATP binding

Biological Process: spermatogenesis; phosphorylation; protein amino acid phosphorylation

NCBI Summary:There are approximately 40 known eukaryotic LIM proteins, so named for the LIM domains they contain. LIM domains are highly conserved cysteine-rich structures containing 2 zinc fingers. Although zinc fingers usually function by binding to DNA or RNA, the LIM motif probably mediates protein-protein interactions. LIM kinase-1 and LIM kinase-2 belong to a small subfamily with a unique combination of 2 N-terminal LIM motifs and a C-terminal protein kinase domain. The protein encoded by this gene is phosphorylated and activated by ROCK, a downstream effector of Rho, and the encoded protein, in turn, phosphorylates cofilin, inhibiting its actin-depolymerizing activity. It is thought that this pathway contributes to Rho-induced reorganization of the actin cytoskeleton. At least three transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:P53671
NCBI GenInfo Identifier:1708824
NCBI Gene ID:3985
NCBI Accession:P53671.1
UniProt Secondary Accession:P53671,Q7KZ80, Q7L3H5, Q96E10, Q99464, Q9UFU0, A8K6H5
UniProt Related Accession:P53671
Molecular Weight:638
NCBI Full Name:LIM domain kinase 2
NCBI Synonym Full Names:LIM domain kinase 2
NCBI Official Symbol:LIMK2  
NCBI Protein Information:LIM domain kinase 2
UniProt Protein Name:LIM domain kinase 2
Protein Family:LIM domain kinase
UniProt Gene Name:LIMK2  
UniProt Entry Name:LIMK2_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-LIMK1/2 (Phospho-Thr505) Antibody, Anti-LIMK1/2 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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