Anti-Transferrin antibody [RM1092] - Serum Loading Control

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Transferrin antibody [RM1092] - Serum Loading Control (AB316295)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling Transferrin with ab316295 at 1/100 (4.93 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human liver.
The section was incubated with ab316295 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Transferrin antibody [RM1092] - Serum Loading Control (AB316295)

Immunohistochemical analysis of paraffin-embedded Human placenta tissue labeling Transferrin with ab316295 at 1/100 (4.93 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human placenta.
The section was incubated with ab316295 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Transferrin antibody [RM1092] - Serum Loading Control (AB316295)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labelling Transferrin with ab316295 at 1/500 (0.98 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing cytoplasmic staining in HepG2 cell line.
Negative control : K-562
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5ug/ml) dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Transferrin antibody [RM1092] - Serum Loading Control (AB316295)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized K-562 (human chronic myelogenous leukemia lymphoblast, Left) / HepG2 (human hepatocellular carcinoma epithelial cell, Right) cells labelling Transferrin with ab316295 at 1/5000 dilution (0.01 ug)/Red compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Negative control : K-562. For ab316295, positive staining occurred in HepG2 cells and no staining in K-562 cells. The control batch (ab82411) showed similar staining pattern. In addition, ab316295 showed stronger staining than ab82411 in HepG2 cells.

• IP

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Immunoprecipitation - Anti-Transferrin antibody [RM1092] - Serum Loading Control (AB316295)

Transferrin was immunoprecipitated from 0.35 mg HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate with ab316295 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab316295 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 2 : ab316295 IP in HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab316295 in HepG2 whole cell lysate

All lanes:

Immunoprecipitation - Anti-Transferrin antibody [RM1092] - Serum Loading Control (ab316295) at 1/30 dilution

All lanes:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 180s

• WB

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Western blot - Anti-Transferrin antibody [RM1092] - Serum Loading Control (AB316295)

Negative control : K-562.

The lysates were freshly made and used for Western Blotting immediately to minimize protein degradation.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-Transferrin antibody [RM1092] - Serum Loading Control (ab316295) at 1/1000 dilution

Lane 1:

HepG2(human hepatocellar carcinoma epithelial cell) whole cell lysate at 40 µg

Lane 2:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 40 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 77 kDa,36 kDa

false

Exposure time: 15s

• WB

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Western blot - Anti-Transferrin antibody [RM1092] - Serum Loading Control (AB316295)

Transferrin might be easily degraded. The expression molecular weight observed is consistent with what has been described in the literature (PMID : 15271890).

All lanes:

Western blot - Anti-Transferrin antibody [RM1092] - Serum Loading Control (ab316295) at 1/1000 dilution

Lane 1:

Human plasma tissue lysate at 20 µg

Lane 2:

Human testis tissue lysate at 20 µg

Lane 3:

Human liver tissue lysate at 20 µg

Lane 4:

Human skeletal muscle tissue lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 77 kDa

false

Exposure time: 26s