Dysferlin Colorimetric Cell-Based ELISA Kit

(No reviews yet) Write a Review
SKU:
CBCAB00628
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
Frequently bought together:

Description

system_update_altDatasheetMSDS

Dysferlin Colorimetric Cell-Based ELISA Kit

The Dysferlin Colorimetric Cell-Based ELISA Kit from Assay Genie is a powerful tool for the accurate measurement of dysferlin levels in cell lysates and tissue homogenates. This kit offers high sensitivity and specificity, ensuring precise and reliable results for researchers studying dysferlin-related diseases and conditions.Dysferlin is a key protein involved in muscle membrane repair and regeneration, making it a crucial biomarker for muscular dystrophy, myopathy, and other muscle-related disorders.

By accurately measuring dysferlin levels, researchers can gain valuable insights into disease progression, treatment efficacy, and potential therapeutic targets.With its user-friendly protocol and flexible sample compatibility, the Dysferlin Colorimetric Cell-Based ELISA Kit is an essential tool for uncovering the role of dysferlin in health and disease. Trust Assay Genie for high-quality research tools to advance your scientific discoveries.

Product Name:Dysferlin Colorimetric Cell-Based ELISA
Product Code:CBCAB00628
ELISA Type:Cell-Based
Target:Dysferlin
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

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

Qualitative determination of Dysferlin concentration is achieved by an indirect ELISA format. In essence, Dysferlin is captured by Dysferlin-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: 8291, UniProt ID: O75923, OMIM: 253601/254130/603009/606768, Unigene: Hs.252180
Gene Symbol:DYSF
Sub Type:None
UniProt Protein Function:Key calcium ion sensor involved in the Ca2+-triggered synaptic vesicle-plasma membrane fusion. Plays a role in the sarcolemma repair mechanism of both skeletal muscle and cardiomyocytes that permits rapid resealing of membranes disrupted by mechanical stress.
NCBI Summary:The protein encoded by this gene belongs to the ferlin family and is a skeletal muscle protein found associated with the sarcolemma. It is involved in muscle contraction and contains C2 domains that play a role in calcium-mediated membrane fusion events, suggesting that it may be involved in membrane regeneration and repair. In addition, the protein encoded by this gene binds caveolin-3, a skeletal muscle membrane protein which is important in the formation of caveolae. Specific mutations in this gene have been shown to cause autosomal recessive limb girdle muscular dystrophy type 2B (LGMD2B) as well as Miyoshi myopathy. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Aug 2008]
UniProt Code:O75923
NCBI GenInfo Identifier:195976821
NCBI Gene ID:8291
NCBI Accession:NP_001123927.1
UniProt Secondary Accession:O75923,A0FK00, B1PZ70, B1PZ71, B1PZ72, B1PZ73, B1PZ74 B1PZ75, B1PZ76, B1PZ77, B1PZ78, B1PZ79,
UniProt Related Accession:O75923
Molecular Weight:
NCBI Full Name:dysferlin isoform 2
NCBI Synonym Full Names:dysferlin
NCBI Official Symbol:DYSF  
NCBI Official Synonym Symbols:MMD1; FER1L1; LGMD2B  
NCBI Protein Information:dysferlin
UniProt Protein Name:Dysferlin
UniProt Synonym Protein Names:Dystrophy-associated fer-1-like protein; Fer-1-like protein 1
Protein Family:Dysferlin
UniProt Gene Name:DYSF  
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-Dysferlin 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.)

0 Reviews

View AllClose

Related Products