/, Cytokines/Human Fibronectin 1 Recombinant

Human Fibronectin 1 Recombinant

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$70.00$1,500.00

SKU: RKP02751 Tags: , , , ,

Description

Accession
P02751
Source
Optimized DNA sequence encoding fibronectin (LYS2175 - GLU2386) including a C-terminal His tag was expressed in HEK293 cells.
Molecular weight
Recombinant human Fibronectin is a protein consisting of 220 amino acid residue subunits,due to glycosylation migrates as an approximately 28kDa bands protein on reduced SDS-PAGE.
Purity
>95%, as determined by SDS-PAGE and HPLC
Endotoxin
Endotoxin content was assayed using a LAL gel clot method. Endotoxin level was found to be less than 0.1 ng/µg(1EU/µg).
Presentation
Recombinant Human Fibronectin is lyophilized from 0.2 μm filtered PBS solution, pH7.2 , 5% Trehalose.
Reconstitution
A quick spin of the vial followed by reconstitution in distilled water to a concentration not less than 0.1 mg/mL. This solution can then be diluted into other buffers.
Storage
Recombinant Fibronectin can be stored in working aliquots at 2° - 8° C for one month, or at -20°C to-70°Cfor twelve months. Avoid repeated freeze/thaw cycles.
Usage
This product is for research purposes only.It may not be used for therapeutics or diagnostic purposes.
Interactor
O41931
Interactor
P02452
Interactor
O41949
Interactor
Interactor
P05106
Interactor
P14738
Interactor
Interactor
Biological Process
Biological Process
Biological Process
Molecular function

Methods

2.1. Peptides, Antibodies, and Chemicals

  • Human recombinant M-CSF, IL-3, and RANKL were acquired from, human recombinant Fibronectin and Collagenase II were obtained from; Mouse-anti-Human CD68 , FITC labeled Mouse-anti-Human CD14, CD45 , APC-labeled Mouse-anti-Human CD90 , and PE-labeled mouse-anti-human CD105 (southern biotech, USA) antibodies were used; cell culture medium and supplements were provided by , all other chemicals were obtained from(-Aldrich, ).

Immunofluorescence staining

  • Murine T cells: 12 mm round glass coverslips were acid washed with 10% H2O2 in 0.1N HCL, rinsed in ddH2O followed by methanol, and flame dried.
  • Coverslips were coated with 50 μg/ml of human fibronectin for 2 h atT.
  • T cells (1.5 × 105) were resuspended in 50 μl unsupplemented DMEM, added to each coverslip and allowed to settle in a humidified chamber for 30 min at 37°C.
  • Unbound cells were washed off and bound T cells were fixed using 3% PFA/PBS and quenched with 50 mM ammonium chloride.
  • To quantify uropod formation, at least 50 randomly selected cells per coverslip were scored based on an elongated shape and the presence of a distinct tail-like structure.
  • For staining of moesin, cells were permeabilized with 0.1% Triton X-100 after fixation, blocked with 2% horse serum in PBS and probed with rabbit anti-moesin, followed by Alexa Fluor 488 goat-anti-rabbit .
  • Cells were imaged using a Zeiss Axiovert 200M microscope with a 63× objective.
  • ERM polarity…

Immunofluorescence staining

  • Murine T cells: 12 mm round glass coverslips were acid washed with 10% H2O2 in 0.1N HCL, rinsed in ddH2O followed by methanol, and flame dried.
  • Coverslips were coated with 50 μg/ml of human fibronectin for 2 h atT.
  • T cells (1.5 × 105) were resuspended in 50 μl unsupplemented DMEM, added to each coverslip and allowed to settle in a humidified chamber for 30 min at 37°C.
  • Unbound cells were washed off and bound T cells were fixed using 3% PFA/PBS and quenched with 50 mM ammonium chloride.
  • To quantify uropod formation, at least 50 randomly selected cells per coverslip were scored based on an elongated shape and the presence of a distinct tail-like structure.
  • For staining of moesin, cells were permeabilized with 0.1% Triton X-100 after fixation, blocked with 2% horse serum in PBS and probed with rabbit anti-moesin, followed by Alexa Fluor 488 goat-anti-rabbit .
  • Cells were imaged using a Zeiss Axiovert 200M microscope with a 63× objective.
  • ERM polarity…
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