Anti-NEFL/NF-L Antibody Picoband®

Western blot analysis of NEFL/NF-L using anti-NEFL/NF-L antibody (A02482-1).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: human SH-SY5Y whole cell lysates,
Lane 2: rat brain tissue lysates,
Lane 3: mouse brain tissue lysates.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-NEFL/NF-L antigen affinity purified polyclonal antibody (Catalog # A02482-1) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for NEFL/NF-L at approximately 72KD. The expected band size for NEFL/NF-L is at 72KD.

IHC analysis of NF-L/NEFL using anti-NF-L/NEFL antibody (A02482-1).
NF-L/NEFL was detected in a paraffin-embedded section of human brain tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-NF-L/NEFL Antibody (A02482-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

IHC analysis of NEFL/NF-L using anti-NEFL/NF-L antibody (A02482-1).
NEFL/NF-L was detected in paraffin-embedded section of mouse spinal cord tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml rabbit anti-NEFL/NF-L Antibody (A02482-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

IHC analysis of NEFL/NF-L using anti-NEFL/NF-L antibody (A02482-1).
NEFL/NF-L was detected in paraffin-embedded section of rat spinal cord tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml rabbit anti-NEFL/NF-L Antibody (A02482-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

IHC analysis of NEFL/NF-L using anti-NEFL/NF-L antibody (A02482-1).
NEFL/NF-L was detected in paraffin-embedded section of mouse spinal cord tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/ml rabbit anti-NEFL/NF-L Antibody (A02482-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

IHC analysis of NEFL/NF-L using anti-NEFL/NF-L antibody (A02482-1).
NEFL/NF-L was detected in a paraffin-embedded section of Human hippocampus tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-NEFL/NF-L Antibody (A02482-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

IF analysis of NEFL/NF-L using anti-NEFL/NF-L antibody (A02482-1).
NEFL/NF-L was detected in a paraffin-embedded section of mouse brain tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 5 μg/mL rabbit anti-NEFL/NF-L Antibody (A02482-1) overnight at 4°C. Biotin conjugated goat anti-rabbit IgG (BA1003) was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Cy3 Conjugated Avidin (BA1037). The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

IF analysis of NEFL/NF-L using anti-NEFL/NF-L antibody (A02482-1).
NEFL/NF-L was detected in a paraffin-embedded section of rat brain tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 5 μg/mL rabbit anti-NEFL/NF-L Antibody (A02482-1) overnight at 4°C. Biotin conjugated goat anti-rabbit IgG (BA1003) was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Cy3 Conjugated Avidin (BA1037). The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Flow Cytometry analysis of 293T cells using anti-NEFL/NF-L antibody (A02482-1).
Overlay histogram showing 293T cells stained with A02482-1 (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-NEFL/NF-L Antibody (A02482-1, 1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.