MA5-16808

antibody from Invitrogen Antibodies

Targeting: S100A9 60B8AG, CAGB, CFAG, CGLB, LIAG, MAC387, MIF, MRP14, NIF, P14

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunohistochemistry (Recommended by provider)

Flow cytometry (Recommended by provider)

Antibody Data

Product number

MA5-16808

Provider

Invitrogen Antibodies

Product name

Calprotectin Monoclonal Antibody (MAC387)

Antibody type

Monoclonal

Antigen

Other

Description

This product requires protein digestion pre-treatment of paraffin sections using trypsin or pronase. A suggested positive control for immunohistochemical applications is human spleen. For FACS analysis, use 10 µL of the suggested working dilution to label 1x10^6 cells in 100 µL. This product is a low-endotoxin, preservative-free formulation. Endotoxin level is

Reactivity

Human, Rat, Bovine, Canine, Feline, Goat, Guinea Pig, Porcine, Rabbit

Host

Mouse

Isotype

IgG

Antibody clone number

MAC387

Vial size

500 µg

Concentration

1 mg/mL

Storage

-20° C, Avoid Freeze/Thaw Cycles

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-Calprotectin Monoclonal Antibody (MAC387) (Product # MA5-16808) and a 13kDa band corresponding to Calprotectin was observed in SK-BR-3 and THP-1 but was absent in Raji and SH-SY5Y which are reported to be negative. Expression of Calprotectin was downregulated upon treatment of THP-1 with PMA/TPA [10.1073/pnas.0709958105]. Membrane enriched cell extracts (30 µg lysate) of SK-BR-3 (Lane 1), THP-1 (Lane 2), THP-1 treated with PMA/TPA (50ng/ml for 24 hours) (Lane 3), Raji (Lane 4) and SH-SY5Y (Lane 5) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of Calprotectin was performed using log phase THP-1 and Raji cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with Calprotectin Monoclonal Antibody (MAC387) (Product # MA5-16808) at 1:250 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing localization to plasma membrane, nucleus and cytoplasm. Panel e shows Raji cells with no expression of Calprotectin. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.