All lanes : Anti-TM9SF2 antibody [EPR24416-17] (ab271123) at 1/1000 dilution

Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma), whole cell lysate
Lane 2 : HT-29 (human colorectal adenocarcinoma epithelial cell), whole cell lysate
Lane 3 : PANC-1 (human pancreatic epithelioid carcinoma epithelial cell), whole cell lysate
Lane 4 : U-87 MG (human glioblastoma-astrocytoma epithelial cell), whole cell lysate
Lane 5 : Human kidney tissue lysate

Lysates/proteins at 40 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/2000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated; Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG)

Predicted band size: 76 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?

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

Exposure time: 3 minutes

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labelling TM9SF2 with ab271123 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human kidney. The section was incubated with ab271123 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 solution2) for 20 mins

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized U-87 MG (Human glioblastoma-astrocytoma epithelial cell) cells labelling  with ab271123 at 1/50 (Red) compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 was used as the secondary antibody.

All lanes : Anti-TM9SF2 antibody [EPR24416-17] (ab271123) at 1/1000 dilution

Lane 1 : C6 (rat glial tumor glial cell), whole cell lysate
Lane 2 : PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate
Lane 3 : Mouse kidney tissue lysate
Lane 4 : Mouse colon tissue lysate
Lane 5 : Rat colon tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 76 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?

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

Samples are non-boiled as boiling may cause protein aggregates.

Exposure time 3 minutes

Immunohistochemical analysis of paraffin-embedded Mouse pancreas tissue labelling TM9SF2 with ab271123 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse pancreas. The section was incubated with ab271123 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 solution2) for 20 mins

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse kidney tissue labelling TM9SF2 with ab271123 at 1100 (5.93 μg/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (Green). Cytoplasmic staining on mouse kidney is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

All lanes : Anti-TM9SF2 antibody [EPR24416-17] (ab271123) at 1/1000 dilution

Lane 1 : HEK-293T (human embryonic kidney) transfected with an empty vector, containing a myc-His-tag®, whole cell lysate
Lane 2 : HEK-293T transfected with human TM9SF2 expression vector containing a myc-His-tag®, whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 76 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?

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

Band higher than 250 kDa should be TM9SF2 aggregates.

Exposure time: 10 seconds

Immunohistochemical analysis of paraffin-embedded Rat pancreas tissue labelling TM9SF2 with ab271123 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat pancreas. The section was incubated with ab271123for 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 solution2) for 20 mins

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat kidney tissue labelling TM9SF2 with ab271123 at 1100 (5.93 μg/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (Green). Cytoplasmic staining on rat kidney is observed. The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).