Anti-Transferrin antibody [EPR20149-224] - Serum Loading Control

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Transferrin antibody [EPR20149-224] - Serum Loading Control (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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Transferrin antibody [EPR20149-224] - Serum Loading Control (AB277635)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Transferrin antibody [EPR20149-224] - Serum Loading Control (AB277635)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Transferrin antibody [EPR20149-224] - Serum Loading Control (AB277635)

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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Transferrin antibody [EPR20149-224] - Serum Loading Control (AB277635)

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.

• IP

Supplier Data

Immunoprecipitation - Anti-Transferrin antibody [EPR20149-224] - Serum Loading Control (AB277635)

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:

Immunoprecipitation - Anti-Transferrin antibody [EPR20149-224] - Serum Loading Control (ab277635)

Predicted band size: 77 kDa

Observed band size: 77 kDa

false

• WB

Lab

Western blot - Anti-Transferrin antibody [EPR20149-224] - Serum Loading Control (AB277635)

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

This blot was developed using a higher sensitivity ECL substrate.

Exposure time : 3 minutes.

All lanes:

Western blot - Anti-Transferrin antibody [EPR20149-224] - Serum Loading Control (ab277635) at 1/1000 dilution

All lanes:

HepG2 (human hepatocellular carcinoma epithelial cell), whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 77 kDa

Observed band size: 77 kDa

false

• WB

Lab

Western blot - Anti-Transferrin antibody [EPR20149-224] - Serum Loading Control (AB277635)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. Exposure times : Lane 1-4 : 3.25 seconds Lane 5 : 15 seconds.

All lanes:

Western blot - Anti-Transferrin antibody [EPR20149-224] - Serum Loading Control (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)(<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 77 kDa

Observed band size: 77 kDa

false