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    products/cell-lines/human-nrp1-knockout-a549-cell-line-ab269507.pdf

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Cardiovascular Angiogenesis Growth Factors VEGF VEGF Receptors
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Human NRP1 knockout A549 cell line (ab269507)

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Western blot - Human NRP1 knockout A549 cell line (ab269507)

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    Overview

    • Product name

      Human NRP1 knockout A549 cell line
    • Parental Cell Line

      A549
    • Organism

      Human
    • Mutation description

      Knockout achieved by CRISPR/Cas9; X = 1 bp deletion; Frameshift = 99.52%
    • Passage number

      <20
    • Knockout validation

      Next Generation Sequencing (NGS), Western Blot (WB)
    • Tested applications

      Suitable for: WBmore details
    • Biosafety level

      1
    • General notes

      Recommended control: Human wild-type A549 cell line (ab259777). Please note a wild-type cell line is not automatically included with a knockout cell line order, if required please add recommended wild-type cell line at no additional cost using the code WILDTYPE-TMTK1.

      Cryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.

      Culture medium: DMEM:Hams F12 + 5% FBS

      Initial handling guidelines: Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C may result in loss of viability.

      1. Thaw the vial in 37°C water bath for approximately 1-2 minutes.
      2. Transfer the cell suspension (0.8 mL) to a 15 mL/50 mL conical sterile polypropylene centrifuge tube containing 8.4 mL pre-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 mL represents minimum recommended dilution. 20 mL represents maximum recommended dilution.
      3. Resuspend the cell pellet in 5 mL pre-warmed culture medium and count using a haemocytometer or alternative cell counting method. Based on cell count, seed cells in an appropriate cell culture flask at a density of 2x103-1x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.
      4. Incubate the culture at 37°C incubator with 5% CO2. Cultures should be monitored daily.

      Subculture guidelines:

      • All seeding densities should be based on cell counts gained by established methods.
      • A guide seeding density of 6x104 cells/cm2 is recommended.
      • A partial media change 24 hours prior to subculture may be helpful to encourage growth, if required.
      • Cells should be passaged when they have achieved 80-90% confluence.
      • Do not exceed 7x104 cells/cm2.

      This product is subject to limited use licenses from The Broad Institute and ERS Genomics Limited, and is developed with patented technology. For full details of the limited use licenses and relevant patents please refer to our limited use license and patent pages.

    Properties

    • Number of cells

      1 x 106 cells/vial, 1 mL
    • Viability

      ~80%
    • Adherent /Suspension

      Adherent
    • Tissue

      Lung
    • Cell type

      epithelial
    • Disease

      Carcinoma
    • Gender

      Male
    • Mycoplasma free

      Yes
    • Storage instructions

      Shipped on Dry Ice. Store in liquid nitrogen.
    • Storage buffer

      Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether
    • Research areas

      • Cardiovascular
      • Angiogenesis
      • Growth Factors
      • VEGF
      • VEGF Receptors
      • Neuroscience
      • Neurology process
      • Growth and Development
      • Axonal Guidance Proteins
      • Immunology
      • Adaptive Immunity
      • Regulatory T Cells

    Target

    • Function

      The membrane-bound isoform 1 is a receptor involved in the development of the cardiovascular system, in angiogenesis, in the formation of certain neuronal circuits and in organogenesis outside the nervous system. It mediates the chemorepulsant activity of semaphorins. It binds to semaphorin 3A, The PLGF-2 isoform of PGF, The VEGF-165 isoform of VEGF and VEGF-B. Coexpression with KDR results in increased VEGF-165 binding to KDR as well as increased chemotaxis. It may regulate VEGF-induced angiogenesis.
      The soluble isoform 2 binds VEGF-165 and appears to inhibit its binding to cells. It may also induce apoptosis by sequestering VEGF-165. May bind as well various members of the semaphorin family. Its expression has an averse effect on blood vessel number and integrity.
    • Tissue specificity

      The expression of isoforms 1 and 2 does not seem to overlap. Isoform 1 is expressed by the blood vessels of different tissues. In the developing embryo it is found predominantly in the nervous system. In adult tissues, it is highly expressed in heart and placenta; moderately in lung, liver, skeletal muscle, kidney and pancreas; and low in adult brain. Isoform 2 is found in liver hepatocytes, kidney distal and proximal tubules.
    • Sequence similarities

      Belongs to the neuropilin family.
      Contains 2 CUB domains.
      Contains 2 F5/8 type C domains.
      Contains 1 MAM domain.
    • Cellular localization

      Secreted and Cell membrane.
    • Target information above from: UniProt accession O14786 The UniProt Consortium
      The Universal Protein Resource (UniProt) in 2010
      Nucleic Acids Res. 38:D142-D148 (2010) .

      Information by UniProt

    Associated products

    • KO cell lysates

      • Human NRP1 knockout A549 cell lysate (ab269669)
    • Related Products

      • Anti-Neuropilin 1 antibody [EPR3113] - BSA and Azide free (ab184783)

    Applications

    The Abpromise guarantee

    Our Abpromise guarantee covers the use of ab269507 in the following tested applications.

    The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

    Application Abreviews Notes
    WB
    Use at an assay dependent concentration. Predicted molecular weight: 103 kDa.
    Notes
    WB
    Use at an assay dependent concentration. Predicted molecular weight: 103 kDa.

    Images

    • Western blot - Human NRP1 knockout A549 cell line (ab269507)
      Western blot - Human NRP1 knockout A549 cell line (ab269507)
      All lanes : Anti-Neuropilin 1 antibody [EPR3113] (ab81321) at 1/1000 dilution

      Lane 1 : Wild-type A549 cell lysate
      Lane 2 : NRP1 knockout A549 cell lysate
      Lane 3 : MDA-MB-231 cell lysate
      Lane 4 : SK-BR-3 cell lysate

      Lysates/proteins at 20 µg per lane.

      Performed under reducing conditions.

      Predicted band size: 103 kDa
      Observed band size: 125-135 kDa why is the actual band size different from the predicted?



      False colour image of Western blot: Anti-Neuropilin 1 antibody [EPR3113] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab81321 was shown to bind specifically to Neuropilin 1. A band was observed at 125/135 kDa in wild-type A549 cell lysates with no signal observed at this size in NRP1 knockout cell line ab269507 (knockout cell lysate ab269669). To generate this image, wild-type and NRP1 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

    Protocols

    • Hemocytometer protocol
    • Mammalian cell tissue culture techniques protocol

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    References (0)

    Publishing research using ab269507? Please let us know so that we can cite the reference in this datasheet.

    ab269507 has not yet been referenced specifically in any publications.

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