701993

antibody from Invitrogen Antibodies

Targeting: NEFH NF-H, NFH

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Flow cytometry (Supportive data in Antibodypedia)

Antibody Data

Product number

701993

Provider

Invitrogen Antibodies

Product name

NF-H Recombinant Rabbit Monoclonal Antibody (4H6L2)

Antibody type

Monoclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with Monkey, Cat and Bovine.

Antibody clone number

4H6L2

Concentration

0.5 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on whole cell extracts (30 µg lysate) of SH-SY5Y (Lane1), IMR32 (Lane 2), Rat Brain (Lane 3), Mouse Brain (Lane 4) and Mouse cerebellum (Lane 5). The blots were probed with Anti-Neurofilament H/NF-H, Phosphorylated Recombinant Rabbit Monoclonal Antibody (Product # 701993, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 180 and 220 kDa band corresponding to Neurofilament-H was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

For immunofluorescence analysis, retinoic acid (10 um, 5 days) + BDNF (50 ng, 3 days) treated SH-SY5Y cells were fixed and permeabilized for detection of endogenous Neurofilament-H using Anti-Neurofilament H/NF-H, Phosphorylated Recombinant Rabbit Monoclonal Antibody (Product # 701993, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of Neurofilament-H protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating membrane and cytoplasmic localization of Neurofilament-H. Panel e) shows untreated cells with no signal. Panel f) represents control cells with no primary antibody to assess background. The images were captured at 40X magnification.

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Flow Cytometry analysis of endogenous Neurofilament-H was performed on SH-SY5Y cells (untreated, red histogram) and SH-SY5Y cells treated with 10 uM Retinoic acid for 72 hours (blue histogram), labeled with ABfinity™ Anti-Neurofilament H / NF-H, Phosphorylated Recombinant Rabbit Monoclonal Antibody (Product# 701993, 5 ug/ 1M cells) or with rabbit isotype control at 0.5 ug/ml (pink histogram) and detected with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, (Alexa Fluor® 488 conjugate, Product# A27034, 0.4 ug/ml, 1:2500). The purple histogram represents unstained control cells. A representative of 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer (4468770).