Anti-DDX6 antibody [EPR27016-10]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DDX6 antibody [EPR27016-10] (AB307418)

Immunohistochemical analysis of paraffin-embedded (A) Wild-type HAP1 ( tissue lABeling DDX6 with ab307418 at 1/2000 (5.17 ug/ml) followed by a ready to use LeicaDS9800 (Bond? Polymer Refine Detection) was used. Positive staining in (A) wild-type HAP1 cell pellet and no staining in (B) DDX6 knockout HAP1 cell pellet. The section was incubated with ab307418 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) was used. Heat mediated antigen retrieval 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-DDX6 antibody [EPR27016-10] (AB307418)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue lABeling DDX6 with ab307418 at 1/100 (5.17 ug/ml) followed by a ready to use LeicaDS9800 (Bond? Polymer Refine Detection) was used. Positive staining on human tonsil. The section was incubated with ab307418 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) was used. 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-DDX6 antibody [EPR27016-10] (AB307418)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeABilized DDX6 KO HAP1 (DDX6 knockout human chronic myelogenous leukemia near-haploid cell) cells lABelling DDX6 with ab307418 at 1/100 (5.17 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 no staining in DDX6 knockout HAP1 cells and showing punctate cytoplasmic staining in wildtype HAP1 cells.Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor? 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). 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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DDX6 antibody [EPR27016-10] (AB307418)

Immunohistochemical analysis of paraffin-embedded Human liver tissue lABeling DDX6 with ab307418 at 1/100 (5.17 ug/ml) followed by a ready to use LeicaDS9800 (Bond? Polymer Refine Detection) was used. Positive staining on human liver. The section was incubated with ab307418 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) was used. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IP

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Immunoprecipitation - Anti-DDX6 antibody [EPR27016-10] (AB307418)

DDX6 was immunoprecipitated from 0.35 mg Parental HAP1 (wildtype control human chronic myelogenous leukemia near-haploid cell line) whole cell lysate 10 ug with ab307418 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab307418 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : Parental HAP1 (wildtype control human chronic myelogenous leukemia near-haploid cell line) whole cell lysate 10 ug
Lane 2 : ab307418 IP in Parental HAP1 whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab307418 in parental HAP1whole cell lysate

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

Exposure time : 137 seconds

All lanes:

Immunoprecipitation - Anti-DDX6 antibody [EPR27016-10] (ab307418) at 1/1000 dilution

Lane 1:

Parental HAP1 (wildtype control human chronic myelogenous leukemia near-haploid cell line) whole cell lysate 10 μg

Lane 2:

Parental HAP1 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

Observed band size: 54 kDa

false

Exposure time: 137s

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-DDX6 antibody [EPR27016-10] (AB307418)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeABilized PC-12 (rat adrenal gland pheochromocytoma cell) cells lABelling DDX6 with ab307418 at 1/100 (5.17 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 punctate cytoplasmic staining in PC-12 cell line.Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor? 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-DDX6 antibody [EPR27016-10] (AB307418)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeABilized C2C12 (mouse myoblast) cells lABelling DDX6 with ab307418 at 1/100 (5.17 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 punctate cytoplasmic staining in C2C12 cell line.Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor? 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). 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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DDX6 antibody [EPR27016-10] (AB307418)

Immunohistochemical analysis of paraffin-embedded Rat spleen tissue lABeling DDX6 with ab307418 at 1/100 (5.17 ug/ml) followed by a ready to use LeicaDS9800 (Bond? Polymer Refine Detection) was used. Positive staining on rat spleen. The section was incubated with ab307418 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) was used. Heat mediated antigen retrieval 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-DDX6 antibody [EPR27016-10] (AB307418)

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue lABeling DDX6 with ab307418 at 1/100 (5.17 ug/ml) followed by a ready to use LeicaDS9800 (Bond? Polymer Refine Detection) was used. Positive staining on mouse spleen. The section was incubated with ab307418 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) was used. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• WB

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Western blot - Anti-DDX6 antibody [EPR27016-10] (AB307418)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Exposure time : Lanes 1-3 : 15 seconds; Lane 4 : 26 seconds; Lanes 5-6 : 180 seconds.

All lanes:

Western blot - Anti-DDX6 antibody [EPR27016-10] (ab307418) at 1/1000 dilution

All lanes:

Human liver tissue lysate 20 μg

Secondary

All lanes:

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

Observed band size: 54 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-DDX6 antibody [EPR27016-10] (AB307418)

Blocking and diluting buffer and concentration : Intercept? (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBSThe samples were run on a Bis-Tris gel. Performed under reducing conditions. False colour image of Western blot : Anti-DDX6 antibody [EPR27016-10] (ab307418) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab307418 was shown to bind specifically to DDX6. A band was observed at 54 kDa in wild-type HAP1 cell lysates whereas no signal observed at this size in DDX6 knockout cell line. To generate this image, wild-type and DDX6 knockout HAP1 cell lysates were analyzed. First, samples were run on a SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in in Intercept? (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS before incubation with primary antibodies overnight at 4 ЎгC. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye? 800CW) preabsorbed (ab216773) at 1/10000 dilution and Goat anti-Mouse IgG H&L (IRDye? 680RD) preabsorbed (ab216776) at 1/20000 dilution. Exposure time :

All lanes:

Western blot - Anti-DDX6 antibody [EPR27016-10] (ab307418) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate 20 μg

Lane 2:

DDX6 knockout HAP1 whole cell lysate 20 μg

Lane 3:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate 20 μg

Secondary

Lanes 1 - 3:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Lanes 1 - 3:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Observed band size: 54 kDa

false

• WB

Supplier Data

Western blot - Anti-DDX6 antibody [EPR27016-10] (AB307418)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Exposure time :

All lanes:

Western blot - Anti-DDX6 antibody [EPR27016-10] (ab307418) at 1/1000 dilution

Lane 1:

RAW 264.7 (mouse elson murine leukemia virus-induced tumor macrophage) whole cell lysate 20 μg

Lane 2:

PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate 20 μg

Lane 3:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 20 μg

Lane 4:

C2C12 (mouse myoblast) whole cell lysate 20 μg

Lane 5:

Mouse spleen tissue lysate 20 μg

Lane 6:

Rat spleen tissue lysate 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: 54 kDa

false

Exposure time: 15s