ER65439

NF-L Rabbit Polyclonal Antibody

cat.: ER65439

Product Type:	Rabbit polyclonal IgG, primary antibodies
Species reactivity:	Human, Mouse, Rat
Applications:	WB, IF-Cell, IHC-P, FC
Clonality:	Polyclonal

Form:	Liquid
Storage condition:	Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Storage buffer:	PBS (pH7.4), 0.1% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration:	1ug/ul
Purification:	Protein A affinity purified.
Molecular weight:	Predicted band size: 62 kDa
Isotype:	IgG
Immunogen:	Recombinant protein within human NF-L aa 1-543 / 543.
Positive control:	Rat brain tissue lysate, mouse hippocampus tissue lysate, rat hippocampus tissue lysate, human brain tissue lysate, SH-SY5Y, human cerebellum tissue, mouse cerebellum tissue, rat cerebellum tissue.
Subcellular location:	Cell projection, axon, Cytoplasm, cytoskeleton.
Recommended Dilutions: WB IF-Cell IHC-P FC	1:100,000 1:500 1:400-1:2,000 1:1,000
Uniprot #:	SwissProt: P07196 Human \| P08551 Mouse \| P19527 Rat
Alternative names:	68 kDa neurofilament protein 68kDa Neurofilament 68kDa neurofilament protein CMT1F CMT2E FLJ53642 Light molecular weight neurofilament protein NEFL Neurofilament light Neurofilament light polypeptide 68kDa Neurofilament light polypeptide Neurofilament protein, light chain Neurofilament subunit NF L Neurofilament triplet L protein NF-L NF68 NFL NFL_HUMAN

Images

Fig1: Western blot analysis of NF-L on different lysates with Rabbit anti-NF-L antibody (ER65439) at 1/100,000 dilution.

Lane 1: Rat brain tissue lysate
Lane 2: Mouse hippocampus tissue lysate
Lane 3: Rat hippocampus tissue lysate
Lane 4: Human brain tissue lysate

Lysates/proteins at 5 µg/Lane.

Predicted band size: 62 kDa
Observed band size: 68 kDa

Exposure time: 3 seconds;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ER65439) at 1/100,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Fig2: Immunocytochemistry analysis of SH-SY5Y cells labeling NF-L with Rabbit anti-NF-L antibody (ER65439) at 1/500 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-NF-L antibody (ER65439) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

	Fig3: Immunohistochemical analysis of paraffin-embedded human cerebellum tissue with Rabbit anti-NF-L antibody (ER65439) at 1/2,000 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ER65439) at 1/2,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
	Fig4: Immunohistochemical analysis of paraffin-embedded mouse cerebellum tissue with Rabbit anti-NF-L antibody (ER65439) at 1/400 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ER65439) at 1/400 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
	Fig5: Immunohistochemical analysis of paraffin-embedded rat cerebellum tissue with Rabbit anti-NF-L antibody (ER65439) at 1/2,000 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ER65439) at 1/2,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Fig6: Flow cytometric analysis of SH-SY5Y cells labeling NF-L.

Cells were fixed and permeabilized. Then stained with the primary antibody (ER65439, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.