Anti-Gentamicin antibody [EPR27991-65]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Gentamicin antibody [EPR27991-65] (AB314126)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling Gentamicin with ab314126 at 1/500 (1.018 µg/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : No staining on human cerebrum. The section was incubated with ab314126 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-Gentamicin antibody [EPR27991-65] (AB314126)

Gentamicin was immunoprecipitated from 0.35 mg RAW264.7 treated with 0.5mg/mL Gentamicin-crosslink-BSA for 4 hours whole cell lysate with ab314126 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab314126 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : RAW264.7 treated with 0.5mg/mL Gentamicin-crosslink-BSA for 4 hours whole cell lysate Lane 2 : ab314126 IP in RAW264.7 treated with 0.5mg/mL Gentamicin-crosslink-BSA for 4 hours whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab314126 in RAW264.7 treated with 0.5 mg/mL Gentamicin-crosslink-BSA for 4 hours whole cell lysate. Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 3 seconds

All lanes:

Immunoprecipitation - Anti-Gentamicin antibody [EPR27991-65] (ab314126) at 1/30 dilution

All lanes:

RAW264.7 treated with 0.5mg/mL Gentamicin-crosslink-BSA for 4 hours 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: 3s

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Gentamicin antibody [EPR27991-65] (AB314126)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Isotype (Left) / RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 0.5 mg/ml Gentamicin-crosslink-BSA-FITC for 4 hours for 4h (Middle) / RAW 264.7 treated with 0.5 mg/ml crosslink-BSA-FITC for 4h (Right) cells labelling Gentamicin with ab314126 at 1/500 dilution (0.1 ug)/ Middle and 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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Gentamicin antibody [EPR27991-65] (AB314126)

Immunohistochemical analysis of paraffin-embedded RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) labeling Gentamicin with ab314126 at 1/500 (1.018 µg/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Panel A. Positive staining on RAW 264.7 treated with 0.5 mg/mL Gentamicin-crosslink-BSA-FITC for 4h; Panel B. Negligible staining on untreated RAW 264.7. The section was incubated with ab314126 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-Gentamicin antibody [EPR27991-65] (AB314126)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling Gentamicin with ab314126 at 1/100 (5.09 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 µg/ml) dilution (Red). Confocal image showing positive staining in RAW 264.7 cells treated with 0.5mg/mL Gentamicin-crosslink-BSA-FITC for 4 hours, and no staining when 0.5 mg/mL crosslink-BSA-FITC was incubated for 4 hours. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). 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 µg/ml) dilution.

• WB

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Western blot - Anti-Gentamicin antibody [EPR27991-65] (AB314126)

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-Bovine Serum Albumin antibody [EPR12774] (ab192603) staining at 1/1000 dilution. Exposure time : 3 seconds

All lanes:

Western blot - Anti-Gentamicin antibody [EPR27991-65] (ab314126) at 1/1000 dilution

Lane 1:

Untreated RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 2:

RAW264.7 treated with 0.5mg/mL Gentamicin-crosslink-BSA for 4 hours 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 1/100000 dilution

false

Exposure time: 3s

• Dot

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Dot Blot - Anti-Gentamicin antibody [EPR27991-65] (AB314126)

Dot blot analysis of Gentamicin using ab314126 at 1 : 1000 (0.509 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution. Exposure time : 180 seconds. Blocking and diluting buffer and concentration : 5% NFDM/TBST

• cELISA

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Competitive ELISA - Anti-Gentamicin antibody [EPR27991-65] (AB314126)

Indirect ELISA analysis of ab314126 at 20 ng/ml. The Secondary antibody used was Streptavidin-Horseradish Peroxidase at 1 : 20000 dilution. Antigen : Gentamicin. Antigen concentration : 0-25 ng/ml