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LMX1B Colorimetric Cell-Based ELISA

SKU:
CBCAB00943
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
$719
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Description

system_update_altDatasheetMSDS

LMX1B Colorimetric Cell-Based ELISA

The LMX1B Colorimetric Cell-Based ELISA Kit is specifically designed for the quantitative measurement of LMX1B levels in cell lysates, serum, and other biological samples. This kit offers high sensitivity and accuracy, providing researchers with reliable and reproducible results for their studies.LMX1B is a critical transcription factor involved in the regulation of gene expression and is known to play a key role in various biological processes, including development, cell differentiation, and disease pathogenesis. Aberrant expression of LMX1B has been implicated in a range of disorders, such as Parkinson's disease and certain types of cancer, highlighting its significance as a potential biomarker for disease diagnostics and therapeutic development.

With its user-friendly protocol and robust performance, the LMX1B Colorimetric Cell-Based ELISA Kit is an invaluable tool for researchers looking to explore the functional role of LMX1B in biological processes and diseases, ultimately contributing to the advancement of scientific knowledge and clinical research.

Product Name:LMX1B Colorimetric Cell-Based ELISA
Product Code:CBCAB00943
ELISA Type:Cell-Based
Target:LMX1B
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The LMX1B Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect LMX1B protein expression profile in cells. The kit can be used for measuring the relative amounts of LMX1B in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on LMX1B.

Qualitative determination of LMX1B concentration is achieved by an indirect ELISA format. In essence, LMX1B is captured by LMX1B-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: 4010, UniProt ID: O60663, OMIM: 161200/602575, Unigene: Hs.129133
Gene Symbol:LMX1B
Sub Type:None
UniProt Protein Function:LMX1B: Essential for the specification of dorsal limb fate at both the zeugopodal and autopodal levels. Defects in LMX1B are the cause of nail-patella syndrome (NPS); also known as onychoosteodysplasia. NPS is a disease that cause abnormal skeletal patterning and renal dysplasia. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Cell development/differentiation; DNA-binding; Transcription factor

Chromosomal Location of Human Ortholog: 9q33.3

Cellular Component: nucleus

Molecular Function:protein binding; zinc ion binding; sequence-specific DNA binding; transcription factor activity

Biological Process: collagen fibril organization; transcription, DNA-dependent; in utero embryonic development; multicellular organismal development; neuron migration; cerebellum morphogenesis; limb morphogenesis; neuron differentiation; cell proliferation; organ growth; regulation of transcription, DNA-dependent; dorsal/ventral pattern formation; midbrain development; positive regulation of transcription from RNA polymerase II promoter; central nervous system neuron development

Disease: Nail-patella Syndrome

NCBI Summary:This gene encodes a member of LIM-homeodomain family of proteins containing two N-terminal zinc-binding LIM domains, 1 homeodomain, and a C-terminal glutamine-rich domain. It functions as a transcription factor, and is essential for the normal development of dorsal limb structures, the glomerular basement membrane, the anterior segment of the eye, and dopaminergic and serotonergic neurons. Mutations in this gene are associated with nail-patella syndrome. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Mar 2010]
UniProt Code:O60663
NCBI GenInfo Identifier:485956425
NCBI Gene ID:4010
NCBI Accession:O60663.3
UniProt Secondary Accession:O60663,O75463, Q5JU95, Q6ISC9, F8W7W6,
UniProt Related Accession:O60663
Molecular Weight:402
NCBI Full Name:LIM homeobox transcription factor 1-beta
NCBI Synonym Full Names:LIM homeobox transcription factor 1, beta
NCBI Official Symbol:LMX1B  
NCBI Official Synonym Symbols:NPS1; LMX1.2  
NCBI Protein Information:LIM homeobox transcription factor 1-beta; LMX-1.2; LIM/homeobox protein 1.2; LIM/homeobox protein LMX1B
UniProt Protein Name:LIM homeobox transcription factor 1-beta
UniProt Synonym Protein Names:LIM/homeobox protein 1.2; LMX-1.2; LIM/homeobox protein LMX1B
Protein Family:LIM/homeobox protein
UniProt Gene Name:LMX1B  
UniProt Entry Name:LMX1B_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-LMX1B 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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