Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [1]
- Immunocytochemistry [1]
- Flow cytometry [1]
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Validation data
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- Product number
- 701993 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- NF-H Recombinant Rabbit Monoclonal Antibody (4H6L2)
- Antibody type
- Monoclonal
- Antigen
- Synthetic peptide
- Reactivity
- Human, Mouse, Rat
- Host
- Rabbit
- Isotype
- IgG
- Antibody clone number
- 4H6L2
- Vial size
- 100 µg
- Concentration
- 0.5 mg/mL
- Storage
- Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.
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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).
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.
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).