Anti-D Box Binding Protein antibody [EPR22370-51]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-D Box Binding Protein antibody [EPR22370-51] (AB222112)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HEK-293T (human embryonic kidney epithelial cell) cells labelling D Box Binding Protein with ab222112 at 1/100 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in HEK-293T cells transfected with myc-tagged D Box Binding Protein expression vector. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 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 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-D Box Binding Protein antibody [EPR22370-51] (AB222112)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HEK-293T (Human embryonic kidney epithelial cell) (transfected with myc-his tagged D Box Binding Protein expression vector) cells labelling D Box Binding Protein with ab222112 at 1/600 dilution (Right) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Left). Secondary antibody isGoat anti rabbit IgG (Alexa Fluor^® 647, ab150079) at 1/2000 dilution.

Cells were stained with rabbit IgG (Left) or ab222112 (Right). Then stained with anti-myc tag conjugated to Alexa Fluor^® 488.

• IP

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Immunoprecipitation - Anti-D Box Binding Protein antibody [EPR22370-51] (AB222112)

D Box Binding Protein was immunoprecipitated from 0.35 mg HEK-293T (Human embryonic kidney epithelial cell) transfected with myc-His tagged D Box Binding Protein expression vector, whole cell lysate, with ab222112 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab222112 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : HEK-293T (Human embryonic kidney epithelial cell) transfected with myc-His tagged D Box Binding Protein expression vector whole cell lysate 10ug.

Lane 2 : ab222112 IP in HEK-293T transfected with myc-His tagged D Box Binding Protein expression vector whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab222112 in HEK-293T transfected with myc-His tagged D Box Binding Protein expression vector whole cell lysate.

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

Exposure time : 5 seconds.

All lanes:

Immunoprecipitation - Anti-D Box Binding Protein antibody [EPR22370-51] (ab222112)

Predicted band size: 34 kDa

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• WB

Lab

Western blot - Anti-D Box Binding Protein antibody [EPR22370-51] (AB222112)

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

The DBP protein is ubiquitously expressed but mainly at suprachiasmatic nuclei in the hypothalamus or liver, and fine-tuned by circadian clock. The antibody detects a band around the reported molecular weight of DBP in human hypothalamus and brain tissue lysates. Negative control : testis (PMID : 2331750)

Exposure time : 3 minutes.

All lanes:

Western blot - Anti-D Box Binding Protein antibody [EPR22370-51] (ab222112) at 1/1000 dilution

Lane 1:

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

Lane 2:

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

Lane 3:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Human liver lysate at 20 µg

Lane 5:

Human hypothalamus lysate at 20 µg

Lane 6:

Human brain lysate at 20 µg

Lane 7:

Human testis 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

Predicted band size: 34 kDa

Observed band size: 45 kDa

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• WB

Lab

Western blot - Anti-D Box Binding Protein antibody [EPR22370-51] (AB222112)

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

Exposure time : 6 seconds.

All lanes:

Western blot - Anti-D Box Binding Protein antibody [EPR22370-51] (ab222112) at 1/1000 dilution

Lane 1:

HEK-293T (human embryonic kidney) transfected with an empty vector (vector control), containing a myc-His-tag®, whole cell lysate at 10 µg

Lane 2:

HEK-293T transfected with D Box Binding Protein (WT) expression vector containing a myc-His-tag®, whole cell lysate at 10 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

Predicted band size: 34 kDa

Observed band size: 52 kDa

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