Human Fibronectin 1 Recombinant

Human Fibronectin 1 Recombinant

$160.00$2,750.00

SKU: RKP02751 Category: Tags: , , , ,

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 P01730 CD4_HUMAN
Interactor P05106
Interactor P14738
Interactor P21980 TGM2_HUMAN
Interactor P04085 PDGFA_HUMAN
Biological Process Acute-phase
Biological Process Angiogenesis
Biological Process Cell-adhesion
Molecular function Heparin-binding

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…
  • 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 was scored based on ERM localization in the uropod of polarized cells, or to an area less than one third of the cell circumference of unpolarized cells.

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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…
  • 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 was scored based on ERM localization in the uropod of polarized cells, or to an area less than one third of the cell circumference of unpolarized cells.

Read more