Skip to main content

NG2/MCSP Antibody (LHM 2)

Catalog # NB100-2688 | Novus Biologicals a Bio-Techne Brand
Catalog #
Availability
Size / Price
Qty
Loading...
NB100-2688
NB100-2688SS

Key Product Details

Validated by

Knockout/Knockdown

Species Reactivity

Human

Applications

Flow (Cell Surface), Flow Cytometry, Immunocytochemistry/ Immunofluorescence, Immunohistochemistry, Immunohistochemistry-Frozen, Immunohistochemistry-Paraffin, Immunoprecipitation, Knockout Validated, Western Blot

Label

Unconjugated

Antibody Source

Monoclonal Mouse IgG1 kappa Clone # LHM 2

Concentration

1.0 mg/ml

Product Summary for NG2/MCSP Antibody (LHM 2)

Immunogen

A 375P cells crude extract

Localization

Cell Membrane

Clonality

Monoclonal

Host

Mouse

Isotype

IgG1 kappa

Scientific Data Images for NG2/MCSP Antibody (LHM 2)

Knockout Validated: NG2/MCSP Antibody (LHM 2) [NB100-2688] - Western blot shows lysates of HeLa human cervical epithelial carcinoma parental cell line and NG2/MCSP (LHM2) knockout (KO) HeLa cell line. PVDF membrane was probed with 1.0 ug/ml of Mouse Anti-Human NG2/MCSP (LHM2) Polyclonal Antibody (Catalog # NB100-2688) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog #HAF018). Specific band was detected for NG2/MCSP (LHM2) at approximately 300 kDa (as indicated) in the parental HeLa cell line, but is not detectable in the knockout HeLa cell line. This experiment was conducted under reducing conditions.
Western Blot: NG2/MCSP Antibody (LHM 2) [NB100-2688] - Analysis of NG2 expression in HeLa whole cell lysate.
Immunohistochemistry-Paraffin: NG2/MCSP Antibody (LHM 2) [NB100-2688] - Staining of NG2 in human brain using DAB with hematoxylin counterstain.

Applications for NG2/MCSP Antibody (LHM 2)

Application
Recommended Usage

Flow (Cell Surface)

1 - 2.5 ug/mL

Flow Cytometry

1 ug/ml

Immunocytochemistry/ Immunofluorescence

1:100

Immunohistochemistry

1:200

Immunohistochemistry-Frozen

1:200

Immunohistochemistry-Paraffin

1:200

Immunoprecipitation

1:10 - 1:100

Western Blot

1:1000
Application Notes
In WB a band can be seen at ~300 kDa.
Please Note: Optimal dilutions of this antibody should be experimentally determined.

Published Applications

Read 12 publications using NB100-2688 in the following applications:

Formulation, Preparation, and Storage

Purification

Protein G purified

Formulation

Tris-Glycine, 0.15 M NaCl

Preservative

0.05% 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: NG2/MCSP

NG2 (Neuron Glial Antigen 2), also known as MCSP, and CSPG4 is a chondroitin surface proteoglycan. Chondroitin surface proteoglycans play a role in stabilizing cell-substratum interactions during early events of cell spreading. At large, proteoglycans are important for cell proliferation and migration, and may act as signal transducers through C-terminal cytoplasmic binding to scaffolding or signaling proteins. The NG2 glycoprotein is a type I membrane protein expressed in the developing and adult central nervous system (CNS) by subpopulations of glia including oligodendroglial precursor cells (OPCs), and in the developing CNS additionally by pericytes. NG2 is located at the apical cell membrane, however it has also been shown to localize to the lamellipodia of astrocytoma upon phosphorylation by PRKCA. Research has found that NG2 may inhibit neuronal growth (PMC1571583), and also acts as a proapoptotic receptor.

Long Name

Chondroitin Sulfate Proteoglycan NG2/Melanoma Associated Chondroitin Sulfate Proteoglycan

Alternate Names

AN2, CSPG4, HMW-MAA, MCSP, MCSPG, MEL-CSPG

Entrez Gene IDs

1464 (Human)

Gene Symbol

CSPG4

UniProt

Product Documents for NG2/MCSP Antibody (LHM 2)

Certificate of Analysis

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

Product Specific Notices for NG2/MCSP Antibody (LHM 2)

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.

Loading...

Protocols

View specific protocols for NG2/MCSP Antibody (LHM 2) (NB100-2688):

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.

Immunocytochemistry Protocol

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

1. Remove culture medium and wash the cells briefly in PBS. Add 10% formalin to the dish and fix at room temperature for 10 minutes.
2. Remove the formalin and wash the cells in PBS.
3. Permeablize the cells with 0.1% Triton X100 or other suitable detergent for 10 min.
4. Remove the permeablization buffer and wash three times for 10 minutes each in PBS. Be sure to not let the specimen dry out.
5. To block nonspecific antibody binding, incubate in 10% normal goat serum from 1 hour to overnight at room temperature.
6. Add primary antibody at appropriate dilution and incubate overnight at 4C.
7. Remove primary antibody and replace with PBS. Wash three times for 10 minutes each.
8. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
9. Remove secondary antibody and replace with PBS. Wash three times for 10 minutes each.
10. Counter stain DNA with DAPi if required.
Western Blot Protocol

1. Perform SDS-PAGE on samples to be analyzed, loading 10-25 ug of total protein per lane.
2. Transfer proteins to PVDF membrane according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
3. Stain the membrane with Ponceau S (or similar product) to assess transfer success, and mark molecular weight standards where appropriate.
4. Rinse the blot TBS -0.05% Tween 20 (TBST).
5. Block the membrane in 5% Non-fat milk in TBST (blocking buffer) for at least 1 hour.
6. Wash the membrane in TBST three times for 10 minutes each.
7. Dilute primary antibody in 1% Non-fat milk in TBST and incubate overnight at 4C with gentle rocking.
8. Wash the membrane in TBST three times for 10 minutes each.
9. Incubate the membrane in diluted HRP conjugated secondary antibody in blocking buffer (as per manufacturer's instructions) for 1 hour at room temperature.
10. Wash the blot in TBST three times for 10 minutes each (this step can be repeated as required to reduce background).
11. Apply the detection reagent of choice in accordance with the manufacturer's instructions.
Loading...