Overview

  • Product name
    Anti-VEGFC antibody [mAbcam 63221]
    See all VEGFC primary antibodies
  • Description
    Mouse monoclonal [mAbcam 63221] to VEGFC
  • Host species
    Mouse
  • Tested applications
    Suitable for: WB, Flow Cytmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 400 to the C-terminus of Human VEGFC.

    Read Abcam's proprietary immunogen policy (Peptide available as ab97415.)

  • Positive control
    • This antibody gave a positive signal in the following human lysates: Spleen tissue; Lymph Node tissue; Thymus tissue; HepG2 whole cell; HEK293 whole cell. This antibody also detects a band of the expected molecular weight when tested against recombinant VEGFC protein (ab83573).
  • General notes

    This antibody clone is manufactured by Abcam.

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

Properties

Applications

Our Abpromise guarantee covers the use of ab63221 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 a concentration of 10 µg/ml. Detects a band of approximately 47 kDa (predicted molecular weight: 47 kDa).
Flow Cyt Use 1µg for 106 cells.

ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.

 

Target

  • Function
    Growth factor active in angiogenesis, and endothelial cell growth, stimulating their proliferation and migration and also has effects on the permeability of blood vessels. May function in angiogenesis of the venous and lymphatic vascular systems during embryogenesis, and also in the maintenance of differentiated lymphatic endothelium in adults. Binds and activates VEGFR-2 (KDR/FLK1) and VEGFR-3 (FLT4) receptors.
  • Tissue specificity
    Spleen, lymph node, thymus, appendix, bone marrow, heart, placenta, ovary, skeletal muscle, prostate, testis, colon and small intestine and fetal liver, lung and kidney, but not in peripheral blood lymphocyte.
  • Sequence similarities
    Belongs to the PDGF/VEGF growth factor family.
  • Post-translational
    modifications
    Undergoes a complex proteolytic maturation which generates a variety of processed secreted forms with increased activity toward VEGFR-3, but only the fully processed form could activate VEGFR-2. VEGF-C first form an antiparallel homodimer linked by disulfide bonds. Before secretion, a cleavage occurs between Arg-227 and Ser-228 producing an heterotetramer. The next extracellular step of the processing removes the N-terminal propeptide. Finally the mature VEGF-C is composed mostly of two VEGF homology domains (VHDs) bound by non-covalent interactions.
  • Cellular localization
    Secreted.
  • Information by UniProt
  • Database links
  • Alternative names
    • Flt 4L antibody
    • Flt4 ligand antibody
    • FLT4 ligand DHM antibody
    • Flt4-L antibody
    • Flt4L antibody
    • Vascular endothelial growth factor C antibody
    • Vascular endothelial growth factor related protein antibody
    • Vascular endothelial growth factor-related protein antibody
    • VEGF C antibody
    • VEGF-C antibody
    • Vegfc antibody
    • VEGFC_HUMAN antibody
    • VRP antibody
    see all

Images

  • All lanes : Anti-VEGFC antibody [mAbcam 63221] (ab63221) at 10 µg/ml

    Lane 1 : Human spleen tissue lysate - total protein (ab29699)
    Lane 2 : Human lymph node tissue lysate - total protein (ab29871)
    Lane 3 : Human thymus tissue lysate - total protein (ab30146)
    Lane 4 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
    Lane 5 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 47 kDa
    Observed band size: 47 kDa
    Additional bands at: 38 kDa, 55 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 1 minute


    In human spleen, lymph node and thymus tissue lysates, this antibody detects a band close to 47-kDa, corresponding to the predicted MW of VEGF-C. In cell lysates, the antibody detects the same 47-kDa, as well as additional bands around 50-kDa and 37-kDa. The target protein can be glycosylated and the sequence contains both a 31 residue signal sequence and two pro-peptides (residues 32-111 and 228-419). VEGF-C undergoes a complex proteolytic maturation which generates a variety of processed secreted forms.   

    Further characterization on this product is underway. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above.
  • Lane 1 : Anti-VEGFC antibody [mAbcam 63221] (ab63221) at 1 µg/ml
    Lane 2 : Anti-VEGFC antibody [mAbcam 63221] (ab63221) at 10 µg/ml

    All lanes : Recombinant Human VEGFC protein (ab83573)

    Lysates/proteins at 0.1 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 47 kDa
    Observed band size: 47 kDa
    Additional bands at: 38 kDa, 55 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 20 minutes


    Individual chains of ab83573 migrate as a band between 27 and 35 kDa in SDS-PAGE due to post-translational modifications, in particular glycosylation.
  • Overlay histogram showing HepG2 cells stained with ab63221 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab63221, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HepG2 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

References

This product has been referenced in:
  • Deng G  et al. MicroRNA-101 inhibits the migration and invasion of intrahepatic cholangiocarcinoma cells via direct suppression of vascular endothelial growth factor-C. Mol Med Rep 12:7079-85 (2015). Read more (PubMed: 26299768) »
See 1 Publication for this product

Customer reviews and Q&As

1-2 of 2 Abreviews or Q&A

Question

Thanks for reply. Please find enclosed notes to the inquiries you asked and feel free let me know if there is further question.

Thanks

1) Sample: Cell extract

Could you specify the species of the samples and the cell or tissue types?

The human species, gastric cancer cell lines (MKN-45,MKN-28,SGC-7901,NCI-N87,GES-1,BGC).

The experiment purpose is to observe VEGF-C in six cells expression differences.

2) Transfer:

Have the proteins been properly transferred onto the membranes? Did you have a chance to test this with Ponceau dye?

Yes, the proteins had been properly transferred onto the membranes. I could observe by the mark.

3) Positive control:

Positive control - this antibody has been tested and characterized for Western blot application on spleen, lymph node, thymus, HepG2, HEK293 lysate. I would suggest running any of these lysates along with the samples on the same gel.

I agree to your suggestion. And I looked up many related articles, I think the problem may appear that VEGF-C should express in these cells.

4) Loading control:

Any loading control (GAPDH, beta-actin, alpha-tubulin) has been run on the same gel to see of the sample preparation and the loading was appropriate?

I use the GAPDH as loading control, and it has been run on the same gel. I will send you some pictures.

5) Detections ystem:

Does the detection system work fine? Have you used it successfully with another primary antibody?

The system exhibited fine. The abcam is said the best in Antibody world, I have not used another primary antibody for the time being.

Read More
Answer

Thank you for getting back to me and for answering to my further questions.

As you may be aware Our policy is that we will offer pre- and post-sale technical support for products sold via unauthorized distributors. In case of problems with our antibodies we will offer suggestions to the researcher's protocol but we will not offer our guarantee as we cannot control the shipping and storage conditions managed by the unauthorized distributor.

Accoding to the NCBI database, stomach has very low levels VEGFC:

http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Hs.435215

I would advise to run either a purified VEGFC protein on the gel or lysate any of the well established cell lines as positive control (tested for Western blot assay as the datasheet indicates) i.e. Spleen (Human) Tissue Lysate - adult normal tissue (ab29699), Lymph node (Human) Tissue Lysate - adult normal tissue (ab29871), Thymus (Human) Tissue Lysate - adult normal tissue (ab30146), HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate and HEK293 (Human.

I hope this will be useful.

Read More

Question

Dear Technical, would you please take a look over the attached antibody and advise what you may suggest for the problem.

Thanks a lot







Product Information





Product Name: VEGF-C antibody



Cat. # GR29205-1





Lot No: ab63221



Ordered:110211AC-1






. Order details:

1. Antibody storage conditions ( store at -20ºC )

2. Please describe the problem ( no band )



3. On what material are you testing the antibody in WB?


Cell extract


4. The lysate


How much protein was loaded: 50ug



What lysis buffer was used: RIPA

What protease inhibitors were used: PMSF

What loading buffer was used: 4 × loading buffer

Did you heat the samples: temperature and time: 100℃ for 10min




5. Electrophoresis/Gel conditions/ Transfer conditions


Reducing or non reducing gel: SDS-PAGE

Gel percentage : 10%

Transfer conditions: 100V 60min




6. Blocking conditions


Buffer: the antibody buffer

Blocking agent: milk , what percentage: 5%

Incubation time: 1h

Incubation temperature: Room temperature




7. Primary Antibody


Specification (in which species was it raised against): Mouse anti-human



At what dilution(s) have you tested this antibody: 1:100

What dilution buffer was used: 5% non-fat milk

Incubation time: overnight

Incubation temperature:4℃

What washing steps were done: 10 min for three times with TBST




8. Secondary Antibody


Specification (in which species was it rose against)? Goat anti-mouse

At what dilution(s) have you tested this antibody: 5% non-fat milk

Incubation time: 1h

Wash steps: 10 min for three times with TBST

Do you know whether the problems you are experiencing come from the secondary? I do not think the problems are coming from secondary.




9. Detection method
ECl, ECl+, other detection method: ECL



10. Background bands


Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): Yes.

Is the blocking step sufficient? Yes.

Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) Yes.

At what size are the bands migrating? No bands. Could they be degradation products of your target? No.

Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient)




11. Did you apply positive and negative controls along with the samples? Please specify.

12. Optimization attempts


How many times have you tried the Western? 16 times

Do you obtain the same results every time e.g. are background bands always in the same place? There were no bands obtained at all.

What steps have you altered?

Read More
Answer

Thank you for your enquiry regarding ab63221 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is having problems with this antibody.

Though you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem.

1) Sample: Cell extract

Could you specify the species of the samples and the cell or tissue types?

2) Transfer:

Have the proteins been properly transferred onto the membranes? Did you have a chance to test this with Ponceau dye?

3) Positive control:

Positive control - this antibody has been tested and characterized for Western blot application on spleen, lymph node, thymus, HepG2, HEK293 lysate. I would suggest running any of these lysates along with the samples on the same gel.

4) Loading control:

Any loading control (GAPDH, beta-actin, alpha-tubulin) has been run on the same gel to see of the sample preparation and the loading was appropriate?

5) Detections ystem:

Does the detection system work fine? Have you used it successfully with another primary antibody?

I look forward to hearing from you soon.

Read More

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