Anti-RFP antibody [EPR28246-45]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-RFP antibody [EPR28246-45] (AB314225)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized 293T cells transfected with a red fluorescent protein expression vector containing a His-tag (upper right) / 293T cells transfected with an empty vector containing a His tag (low right) cells labelling RFP with ab314225 at 1/500 dilution (0.1 ug)/Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-RFP antibody [EPR28246-45] (AB314225)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T (human embryonic kidney epithelial cell) cells labelling RFP with ab314225 at 1/2000 (0.252 ug/ml) dilution, followed by ab150079 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) antibody at 1/500 (4 ug/ml) dilution (Green). Confocal image showing positive staining in 293T cells transfected with a RFP expression vector containing a Myc-tag. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). Myc-Tag Mouse mAb (Alexa Fluor® 488) was used to counterstain at 1/100 (0.5 ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150079 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) at 1/500 (4 ug/ml) dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RFP antibody [EPR28246-45] (AB314225)

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling RFP with ab314225 at 1/2000 (0.252 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : No staining on human kidney. The section was incubated with ab314225 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-RFP antibody [EPR28246-45] (AB314225)

Immunohistochemical analysis of paraffin-embedded Panel A HEK-293T cells transfected with a Red fluorescent protein expression vector containing a his tag. Panel B HEK-293T cells transfected with empty vector containing a his tag. Tissue labeling RFP with ab314225 at 1/2000 (0.252 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on (A) HEK-293T transfected with a red fluorescent protein expression vector and no staining on (B) HEK-293T transfected with empty vector. The section was incubated with ab314225 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-RFP antibody [EPR28246-45] (AB314225)

Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling RFP with ab314225 at 1/2000 (0.252 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : No staining on rat kidney. The section was incubated with ab314225 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-RFP antibody [EPR28246-45] (AB314225)

Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labeling RFP with ab314225 at 1/2000 (0.252 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : No staining on mouse kidney. The section was incubated with ab314225 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.

• IP

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Immunoprecipitation - Anti-RFP antibody [EPR28246-45] (AB314225)

RFP was immunoprecipitated from 0.35 mg 293T cells transfected with a red fluorescent protein expression vector containing a His-tag, whole cell lysate with ab314225 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab314225 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : 293T cells transfected with a Red fluorescent protein expression vector containing a His-tag, whole cell lysate Lane 2 : ab314225 IP in 293T cells transfected with a Red fluorescent protein expression vector containing a His-tag, whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab314225 in 293T cells transfected with a Red fluorescent protein expression vector containing a His-tag, whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-RFP antibody [EPR28246-45] (ab314225) at 1/30 dilution

All lanes:

293T cells transfected with a Red fluorescent protein expression vector containing a His-tag, 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

• WB

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Western blot - Anti-RFP antibody [EPR28246-45] (AB314225)

Blocking and diluting buffer and concentration : 5% NFDM/TBST In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution. In Western blot, Anti-6X His tag antibody [EPR20547]-CHIP Grade (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-RFP antibody [EPR28246-45] (ab314225) at 1/1000 dilution

Lane 1:

293T cells transfected with an empty vector containing a His tag, whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a Red fluorescent protein expression vector containing a His-tag, whole cell 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 1/20000 dilution

Observed band size: 35 kDa

false

Exposure time: 15s

• WB

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Western blot - Anti-RFP antibody [EPR28246-45] (AB314225)

In Western blot, Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 dilution.

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

ab314225 and ab314226 do not recognize GFP.

All lanes:

Western blot - Anti-RFP antibody [EPR28246-45] (ab314225) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

293T cells transfected with an empty vector whole cell lysate at 20 µg

Lane 3:

293T cells transfected with RFP protein whole cell lysate at 20 µg

Lane 4:

293T cells transfected with GFP tagged IFNA2 protein whole cell lysate at 20 µg

Observed band size: 50 kDa

false

Exposure time: 1s