Recombinant Anti-Transferrin antibody [EPR20149-224] (ab277635)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling Transferrin with ab277635 at 1/100 (5.04 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on human liver. The section was incubated with ab277635 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

ab277635 staining Transferrin in HepG2 cells, with negative expression in K562 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilised with 0.1% Triton x-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab277635 at 1 µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 0.5 µg/ml. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150119, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 647), pre-adsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

This product also work with 100% methanol (5 min) fixation under the same testing conditions.

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labelling Transferrin with ab277635 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). 

A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. 

 

 

Transferrin was immunoprecipitated from 0.35 mg Human liver tissue lysate with ab277635 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab277635 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Human liver tissue lysate 10 ug

Lane 2: ab277635 IP in Human liver tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab277635 in Human liver tissue lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 3 minutes.

 

All lanes : Anti-Transferrin antibody [EPR20149-224] (ab277635) at 1/1000 dilution

Lane 1 : Human plasma at 20 µl
Lane 2 : Human breast tissue lysate at 20 µg
Lane 3 : Human kidney tissue lysate at 20 µg
Lane 4 : Human testis tissue lysate at 20 µg
Lane 5 : Human liver tissue lysate at 20 µg

Secondary
Lane 1 : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution
Lanes 2-5 : VeriBlot for IP secondary antibody(HRP)(ab131366) at 1/1000 dilution

Predicted band size: 77 kDa
Observed band size: 77 kDa

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

Exposure times: Lane 1-4: 3.25 seconds

Lane 5: 15 seconds.

 

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HepG2 cells labelling Transferrin with ab277635 at 1/100 (5.04 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in HepG2 cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluo^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling Transferrin with ab277635 at 1/100 (5.04 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on stromal cells of human testis. The section was incubated with ab277635 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Anti-Transferrin antibody [EPR20149-224] (ab277635) at 1/1000 dilution + HepG2 (human hepatocellular carcinoma epithelial cell), whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 77 kDa
Observed band size: 77 kDa

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

This blot was developed using a higher sensitivity ECL substrate.

Exposure time: 3 minutes.