Recombinant Anti-CD9 antibody [EPR23105-121] (ab236630)

All lanes : Anti-CD9 antibody [EPR23105-121] (ab236630) at 1/1000 dilution

Lane 1 : Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : CD9 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : A549 (Human lung carcinoma cell line) whole cell lysate
Lane 4 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 25 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab236630 observed at 18 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab236630 was shown to react with CD9 in wild-type HeLa cells in Western blot with loss of signal observed in CD9 knockout cell line ab255375 (CD9 knockout cell lysate ab263754). Wild-type HeLa and CD9 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab236630 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HCT 116 (human colorectal carcinoma epithelial cell) cells labelling CD9 with ab236630 at 1/500 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing membranous staining in HCT 116 cell line. Negative control: Raji (PMID: 8921952). 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.

All lanes : Anti-CD9 antibody [EPR23105-121] (ab236630) at 1/1000 dilution

Lane 1 : Human tonsil lysate
Lane 2 : Human colon lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 25 kDa
Observed band size: 22 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST. The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 3275469).  Exposure time: 3 minutes.

All lanes : Anti-CD9 antibody [EPR23105-121] (ab236630) at 1/1000 dilution

Lane 1 : HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate
Lane 2 : Raji (human Burkitts lymphoma b lymphocyte) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 25 kDa
Observed band size: 22 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST This blot was developed using a higher sensitivity ECL substrate.  The molecular weight observed is consistent with what has been described in the literature (PMID: 3275469). 

Negative control: Raji (PMID: 8921952).  Exposure time: 3 minutes

CD9 was immunoprecipitated from 0.35 mg HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate with ab236630 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab236630 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.

Lane 1: HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate 10ug.

Lane 2: ab236630 IP in HCT 116 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab236630 in HCT 116 whole cell lysate.

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

Exposure time: 30 seconds.

 

Flow cytometric analysis of Raji (human Burkitt's lymphoma B lymphocyte, Left) / HCT 116 (human colorectal carcinoma epithelial cell, Right) cells labelling CD9 with ab236630 at 1/50 dilution compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 was used as the secondary antibody.

Negative control: Raji (PMID: 8921952).

Gated on viable cells.

Immunohistochemical analysis of paraffin-embedded Human bladder carcinoma tissue labeling CD9 with ab236630 at 1/100 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human bladder carcinoma. The section was incubated with ab236630 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling CD9 with ab236630 at 1/100 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on platelets of human spleen. The section was incubated with ab236630 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.