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CD31/PECAM-1 Antibody (MEC13.3) - BSA Free

Catalog # NB600-1475 | Novus Biologicals a Bio-Techne Brand

Key Product Details

Species Reactivity

Human, Mouse

Applications

CyTOF-ready, Flow (Cell Surface), Flow Cytometry, Immunocytochemistry/ Immunofluorescence, Immunohistochemistry, Immunohistochemistry-Frozen, Immunoprecipitation, In vitro assay, In vivo assay

Label

Unconjugated

Antibody Source

Monoclonal Rat IgG2a Kappa Clone # MEC13.3

Format

BSA Free

Concentration

1.0 mg/ml

Product Summary for CD31/PECAM-1 Antibody (MEC13.3) - BSA Free

Immunogen

This CD31/PECAM-1 Antibody (MEC13.3) was developed against mouse endothelial cell line T-end.

Reactivity Notes

Mouse (PMID: 7956830) and Human (PMID: 30626719) reactivity reported in scientific literature.

Localization

Membrane; Single-pass type I membrane protein. Cell junction.

Clonality

Monoclonal

Host

Rat

Isotype

IgG2a Kappa

Theoretical MW

82.5 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Scientific Data Images for CD31/PECAM-1 Antibody (MEC13.3) - BSA Free

Immunocytochemistry/Immunofluorescence: CD31/PECAM-1 Antibody (MEC13.3) [NB600-1475] - Mouse MS1 cells were fixed in 4% paraformaldehyde for 10 minutes and permeabilized in 0.05% Triton X-100 in PBS for 5 minutes. The cells were incubated with CD31/PECAM-1 Antibody [MEC13.3] (NB600-1475) at 1ug/ml overnight at 4C and detected with an anti-rat DyLight 488 (Green) at a 1:1000 dilution for 60 minutes. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 100X objective and digitally deconvolved.
Immunohistochemistry-Frozen: CD31/PECAM-1 Antibody (MEC13.3) [NB600-1475] - 4T1 tumor sections were stained for CD31 (green) and CD105 (red).
Flow (Cell Surface): CD31/PECAM-1 Antibody (MEC13.3) [NB600-1475] - A surface stain was performed on WEHI-3 Cells with CD31/PECAM-1 Antibody (MEC13.3) (NB600-1475, blue) and a matched isotype control (orange). Cells were incubated in an antibody dilution of 2.5 ug/mL for 20 minutes at room temperature, followed by rat F(ab)2 IgG (H+L) APC-conjugated secondary antibody (F0113, R&D Systems).

Applications for CD31/PECAM-1 Antibody (MEC13.3) - BSA Free

Application
Recommended Usage

Flow (Cell Surface)

2.5 ug/ml

Flow Cytometry

2.5 ug/ml

Immunocytochemistry/ Immunofluorescence

1:500 - 1:1000

Immunohistochemistry

1:100-1:500

Immunohistochemistry-Frozen

1:100

Immunoprecipitation

1:10-1:500

In vitro assay

reported in scientific literature (PMID 24647208)

In vivo assay

reported in scientific literature
Application Notes
This CD31/PECAM1 Antibody (MEC13.3) is useful for in vitro and in vivo blocking of CD31-mediated cell-cell interactions. Please Note: NB600-1475 works well on acetone-fixed frozen sections as well as zinc-fixed paraffin-embedded sections. However, it is not recommended for formalin-fixed paraffin-embedded sections due to inconsistent results. This antibody is CyTOF ready.
Please Note: Optimal dilutions of this antibody should be experimentally determined.

Reviewed Applications

Read 2 reviews rated 5 using NB600-1475 in the following applications:

Published Applications

Read 30 publications using NB600-1475 in the following applications:

Formulation, Preparation, and Storage

Purification

Protein G purified

Formulation

PBS

Format

BSA Free

Preservative

0.02% Sodium Azide

Concentration

1.0 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.

Background: CD31/PECAM-1

Cluster of differentiation (CD31), Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1), is a 30kDa type I transmembrane glycoprotein expressed by endothelial cells, platelets, leukocytes and some lymphocytes (e.g., B and T-cell subsets). Structurally, PECAM-1 consists of six extracellular immunoglobulin-like domains which serve to support various functions including homophilic binding, maintaining high expression of PECAM-1 within endothelial intercellular junctions, and supporting endothelial cell migration (1,2). PECAM's homophilic binding underscores its role in vascular barrier integrity and leukocyte trans-endothelial movement. However, PECAM may also engage in heterophilic interactions with molecules such as glycosaminoglycans (GAG), integrin alpha 5 beta 3, CD38 in lymphocytes and CD177/PR3 in neutrophils (2).

PECAM's intracellular cytoplasmic domain consists of a sequence of 118 amino acids and contains serine and tyrosine (also referred to as immunoreceptor tyrosine-based inhibitory motifs-ITIMs) residues, which may be phosphorylated upon cellular stimulation (3). ITIMs are phosphorylated by Src-family kinases and non-Src family kinases (e.g., Csk), leading to a conformational change which supports interactions with Src homology 2 (SH2) domain containing proteins such as protein-tyrosine phosphatase, SHP-2 (1,2). Formation of SHP-2/PECAM-1 complexes induces endothelial cell migration through the dephosphorylation of focal adhesion kinase and regulation of RhoA activity (1). Signaling downstream of ITIM tyrosine phosphorylations also plays a role in PECAM's anti-apoptotic activity, a function which is independent of its interaction with SHP-2. In platelets and leukocytes, phosphorylation of PECAM's cytosolic domain is inhibitory, preventing their activation.

References

1. Lertkiatmongkol, P., Liao, D., Mei, H., Hu, Y., & Newman, P. J. (2016). Endothelial functions of PECAM-1 (CD31). Current Opinion in Hematology. https://doi.org/10.1097/MOH.0000000000000239.Endothelial

2. Privratsky, J. R., & Newman, P. J. (2014). PECAM-1: Regulator of endothelial junctional integrity. Cell and Tissue Research. https://doi.org/10.1007/s00441-013-1779-3

3. Newman, P. J., & Newman, D. K. (2003). Signal transduction pathways mediated by PECAM-1: New roles for an old molecule in platelet and vascular cell biology. Arteriosclerosis, Thrombosis, and Vascular Biology. https://doi.org/10.1161/01.ATV.0000071347.69358.D9

Long Name

Platelet Endothelial Cell Adhesion Molecule 1

Alternate Names

CD31, EndoCAM, PECA1, PECAM-1, PECAM1, MEC 13.3 CD31, MEC 13.3 Clone

Gene Symbol

PECAM1

Product Documents for CD31/PECAM-1 Antibody (MEC13.3) - BSA Free

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot number in the search box below.

Product Specific Notices for CD31/PECAM-1 Antibody (MEC13.3) - BSA Free

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

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Protocols

View specific protocols for CD31/PECAM-1 Antibody (MEC13.3) - BSA Free (NB600-1475):

Protocol for Flow Cytometry Cell Surface Staining
Sample Preparation.
1. Grow cells to 60-85% confluency. Flow cytometry requires between 2 x 105 and 1 x 106 cells for optimal performance.
2. If cells are adherent, harvest gently by washing once with staining buffer and then scraping. Avoid using trypsin as this can disrupt certain epitopes of interest. If enzymatic harvest is required, use Accutase, Collagenase, or TrypLE Express for a less damaging option.
3. Reserve 100 uL for counting, then transfer cell volume into a 15 mL conical tube and centrifuge for 4 minutes at 400 RCF.
a. Count cells using a hemocytometer and a 1:1 trypan blue exclusion stain to determine cell viability before starting the flow protocol. If cells appear blue, do not proceed.
4. Re-suspend cells to a concentration of 1 x 106 cells/mL in staining buffer (NBP2-26247).
5. Aliquot out 100 uL samples in accordance with your experimental samples.

Tip: When cell surface and intracellular staining are required in the same sample, it is advisable that the cell surface staining be performed first since the fixation and permeablization steps might reduce the availability of surface antigens.
Cell surface staining
1. Recommended: Block non-specific interactions using 0.5-1 ug of a species specific Fc-blocking reagent such as an anti-mouse CD16/CD32 antibody (NBP1-27946).
2. Add appropriate amount of each antibody (eg. 1 test or 1 ug per sample, as experimentally determined) to 100 uL of staining buffer (NBP2-26247) per sample (eg. use 1 mL of staining buffer for 10 samples).
3. Mix well and incubate at room temperature in dark for 20 minutes.
4. Add 1-2 mL of staining buffer and centrifuge at 400 RCF for 1 minute and discard supernatant.
5. Wash twice by re-suspending cells in staining buffer (2 mL for tubes or 200 uL for wells) and centrifuging at 400 RCF for 5 minutes. Discard supernatant.
6. Add appropriate amount of secondary antibody (as experimentally determined) to each sample.
7. Incubate at room temperature in dark for 20 minutes.
8. Add 1-2 mL of staining buffer and centrifuge at 400 RCF for 1 minute and discard supernatant.
9. Wash twice by re-suspending cells in staining buffer (2 mL for tubes or 200 uL for wells) and centrifuging at 400 RCF for 5 minutes. Discard supernatant.
10. Resuspend in an appropriate volume of staining buffer (usually 500 uL per sample) and proceed with analysis on your flow cytometer.

[[URL:https://www.novusbio.com/products/cd31-pecam-1-antibody-mec133_nb600-14… Antibody]]
Immunocytochemistry Protocol

Culture cells to appropriate density in 35 mm culture dishes or 6-well plates.

1. Remove culture medium and add 10% formalin to the dish. Fix at room temperature for 30 minutes.
2. Remove the formalin and add ice cold methanol. Incubate for 5-10 minutes.
3. Remove methanol and add washing solution (i.e. PBS). Be sure to not let the specimen dry out. Wash three times for 10 minutes.
4. To block nonspecific antibody binding incubate in 10% normal goat serum from 1 hour to overnight at room temperature.
5. Add primary antibody at appropriate dilution and incubate at room temperature from 2 hours to overnight at room temperature.
6. Remove primary antibody and replace with washing solution. Wash three times for 10 minutes.
7. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
8. Remove antibody and replace with wash solution, then wash for 10 minutes. Add Hoechst 33258 to wash solution at 1:25,0000 and incubate for 10 minutes. Wash a third time for 10 minutes.
9. Cells can be viewed directly after washing. The plates can also be stored in PBS containing Azide covered in Parafilm (TM). Cells can also be cover-slipped using Fluoromount, with appropriate sealing.

*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow safe laboratory procedures.
Immunohistochemistry-Paraffin Embedded Sections

Antigen Unmasking:
Bring slides to a boil in 10 mM sodium citrate buffer (pH 6.0) then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench-top for 30 minutes (keep slides in the sodium citrate buffer at all times).

Staining:
1. Wash sections in deionized water three times for 5 minutes each.
2. Wash sections in PBS for 5 minutes.
3. Block each section with 100-400 ul blocking solution (1% BSA in PBS) for 1 hour at room temperature.
4. Remove blocking solution and add 100-400 ul diluted primary antibody. Incubate overnight at 4 C.
5. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
6. Add 100-400 ul HRP polymer conjugated secondary antibody. Incubate 30 minutes at room temperature.
7. Wash sections three times in wash buffer for 5 minutes each.
8. Add 100-400 ul DAB substrate to each section and monitor staining closely.
9. As soon as the sections develop, immerse slides in deionized water.
10. Counterstain sections in hematoxylin.
11. Wash sections in deionized water two times for 5 minutes each.
12. Dehydrate sections.
13. Mount coverslips.


FAQs for CD31/PECAM-1 Antibody (MEC13.3) - BSA Free

Showing  1 - 2 of 2 FAQs Showing All
  • Q: I like to know that CD31/PECAM1 Antibody (MEC13.3) (NB600-1475), will work with NBF fixed paraffin embedded sample, not zinc fixed.

    A: It looks like NB600-1475 can be used in IHC of acetone-fixed frozen sections and zinc-fixed paraffin embedded sections. However, it is not recommended for formalin-fixed paraffin-embedded sections due to inconsistent results.

  • Q: CD31/PECAM-1 Antibody (MEC13.3) [NB600-1475] was left out at room temperature for 3 days and is not working. Do you think the antibody is degraded?

    A: This antibody is supplied in PBS with 0.05% Sodium Azide and we suggest storing this antibody at 4C while small aliqots can be stored at -20C also. When in lyophillized form, the antibodies can stay stable to some extent at room temperature but in liquid form, they often degrade and thats probably what happened in your case also. We suggest ordering a new stock of antibody so you do not have to waste your valuable time & resources by trying the non-working or degraded antibody. 

Showing  1 - 2 of 2 FAQs Showing All
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