Recombinant Anti-CD9 antibody [EPR27551-92] (ab307085)

All lanes : Anti-CD9 antibody [EPR27551-92] (ab307085) at 1/1000 dilution

Lane 1 : Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : CD9 knockout HeLa whole cell lysate
Lane 3 : MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate
Lane 4 : RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (ab216773) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (ab216776) at 1/10000 dilution

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-CD9 antibody [EPR27551-92] (ab307085) 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, ab307085 was shown to bind specifically to CD9. A band was observed at 23 kDa in wild-type HeLa cell lysates whereas no signal observed at this size in CD9 knockout cell line ab255375 (knockout cell lysate ab263754). To generate this image, wild-type and CD9 knockout HeLa cell lysates were analyzed. First, samples were run on an 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 °C. 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) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

 Exposure time: 

 

All lanes : Anti-CD9 antibody [EPR27551-92] (ab307085) at 1/1000 dilution

Lane 1 : HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate
Lane 2 : K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate
Lane 3 : Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate
Lane 4 : Mouse brain tissue lysate
Lane 5 : Mouse kidney tissue lysate
Lane 6 : C6 (rat glial tumor glial cell) whole cell lysate

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Blocking and diluting buffer and concentration: 5% NFDM/TBSTNegative controls: K-562, Raji (PMID:17407154 ; PMID: 8921952).

 Exposure time: 70 seconds

 

All lanes : Anti-CD9 antibody [EPR27551-92] (ab307085) at 1/1000 dilution

Lane 1 : Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate 20 µg
Lane 2 : CD9 knockout HeLa whole cell lysate 20 µg
Lane 3 : MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate 20 µg
Lane 4 : RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate 20 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (ab216773) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (ab216776) at 1/10000 dilution

Observed band size: 23 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBST

The samples were run on a Bis-Tris gel.

Performed under reducing conditions.

       

False colour image of Western blot: Anti-CD9 antibody [EPR27551-92] (ab307085) 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, ab307085 was shown to bind specifically to CD9. A band was observed at 23 kDa in wild-type HeLa cell lysates whereas no signal observed at this size in CD9 knockout cell line ab255375 (knockout cell lysate ab263754). To generate this image, wild-type and CD9 knockout HeLa cell lysates were analyzed. First, samples were run on an 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 °C. 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) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes : Anti-CD9 antibody [EPR27551-92] (ab307085) at 1/1000 dilution

Lane 1 : HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate 20 µg
Lane 2 : K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate 20 µg
Lane 3 : Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate 20 µg
Lane 4 : Mouse brain tissue lysate 20 µg
Lane 5 : Mouse kidney tissue lysate 20 µg
Lane 6 : C6 (rat glial tumor glial cell) whole cell lysate 20 µg

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Observed band size: 23 kDa why is the actual band size different from the predicted?

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

Negative controls: K-562, Raji (PMID:17407154 ; PMID: 8921952).

Exposure time: 70 seconds

CD9 was immunoprecipitated from 0.35 mg RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate 10 ug with ab307085 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab307085 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate 10 ug

Lane 2: abab307085 IP in RAW 264.7 whole cell lysate

Lane 3:Rabbit monoclonal IgG (ab172730) instead of ab307085 in RAW 264.7 whole cell lysate

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

Exposure time: 10 seconds

Immunohistochemical analysis of paraffin-embedded Rat kidney tissue labeling CD9 with ab307085 at 1/2000 (0.228 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Membranous staining in rat kidney. The section was incubated with ab307085 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

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling CD9 with ab307085 at 1/2000 (0.228 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining in rat cerebrum. The section was incubated with ab307085 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

Immunohistochemical analysis of paraffin-embedded Mouse lung cancer tissue labeling CD9 with ab307085 at 1/2000 (0.228 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Membranous staining in mouse lung cancer. The section was incubated with ab307085 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

Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labeling CD9 with ab307085 at 1/2000 (0.228 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining in mouse kidney. The section was incubated with ab307085 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

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling CD9 with ab307085 at 1/2000 (0.228 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining in mouse cerebrum. The section was incubated with ab307085 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