Anti-LINE-1 ORF1p 抗体 [EPR21844-108]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-LINE-1 ORF1p antibody [EPR21844-108] (AB216324)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized F9 (mouse embryonal carcinoma epithelial cell) cells labeling LINE-1 ORF1p with ab216324 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining in F9 cell line.

Negative control : NIH/3T3 (PMID : 24324740).

The nuclear counter stain is DAPI (blue). Tubulin is detected with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at a 1/200 dilution (red).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-LINE-1 ORF1p antibody [EPR21844-108] (AB216324)

Flow cytometry overlay histogram showing left F9 positive cells and right negative NIH3T3 stained with ab216324 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 mins. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab216324) (1x 10⁶ in 100μl at 1.0μg/ml (1/1980 dilution)) for 30 mins at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/4000 dilution for 30 mins at 22°C

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in F9 Fixed with 80% methanol (5 mins) / permeabilised with 0.1% PBS-Triton X-100 for 15 minss under the same conditions.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LINE-1 ORF1p antibody [EPR21844-108] (AB216324)

Immunohistochemical analysis of paraffin-embedded mouse liver tissue labeling LINE-1 ORF1p with ab216324 at 1/500 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Negative control : No staining on mouse liver (PMID : 24607009) is observed. Counterstained with hematoxylin.
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-LINE-1 ORF1p antibody [EPR21844-108] (AB216324)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast, Left) / F9 (mouse embryonal carcinoma epithelial cell, Right) cell lines labeling LINE-1 ORF1p with ab216324 at 1/500 (red) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

Negative control : NIH/3T3 (Reference for the negative control is PMID : 24324740).

• IHC-Fr

Collaborator

Immunohistochemistry (Frozen sections) - Anti-LINE-1 ORF1p antibody [EPR21844-108] (AB216324)

ab216324 staining LINE-1 ORF1p in Wt and Maelstrom (Mael) null mutant mouse testis by Immunohistochemistry (Frozen sections). Mael is a repressor of LINE-1 transposable elements, so the expression of LINE-1 ORF1p increases in this KO. Tissue was fixed with paraformaldehyde and blocked with 10% serum for 30 minutes at 25°C; Samples were incubated with primary antibody (1/100, 1/500 and 1/1000) for 12 hours at 4°C in 3% Donkey Serum in PBS + 0.05% Triton X. Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150073) (1/1000) was used as the secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LINE-1 ORF1p antibody [EPR21844-108] (AB216324)

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling LINE-1 ORF1p with ab216324 at 1/500 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining on spermatogonia of mouse testis (PMID : 24607009) is observed. Counterstained with hematoxylin.
Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• IP

Supplier Data

Immunoprecipitation - Anti-LINE-1 ORF1p antibody [EPR21844-108] (AB216324)

LINE-1 ORF1p was immunoprecipitated from 0.35 mg F9 (mouse embryonal carcinoma epithelial cell) whole cell lysate with ab216324 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab216324 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5,000 dilution

Lane 1 : F9 whole cell lysate 10 μg (Input).
Lane 2 : ab216324 IP in F9 whole cell lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab216324 in F9 whole cell lysate.

Blocking/Dilution buffer : 5% NFDM/TBST.
Exposure time : 30 seconds.

All lanes:

Immunoprecipitation - Anti-LINE-1 ORF1p antibody [EPR21844-108] (ab216324)

Predicted band size: 44 kDa

Observed band size: 41 kDa,43 kDa

false

• WB

Supplier Data

Western blot - Anti-LINE-1 ORF1p antibody [EPR21844-108] (AB216324)

Blocking/Dilution buffer : 5% NFDM/TBST.

Negative control : NIH/3T3 (PMID : 24324740).

All lanes:

Western blot - Anti-LINE-1 ORF1p antibody [EPR21844-108] (ab216324) at 1/1000 dilution

Lane 1:

F9 (mouse embryonal carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 2:

NIH/3T3 (mouse embryonic fibroblast), whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 41 kDa

Observed band size: 41 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-LINE-1 ORF1p antibody [EPR21844-108] (AB216324)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-LINE-1 ORF1p antibody [EPR21844-108] (ab216324) at 1/1000 dilution

All lanes:

mouse testis lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 41 kDa

Observed band size: 41 kDa

false

Exposure time: 3min