All lanes : Anti-TVA antibody [PICA187E] (ab271292) at 1/5000 dilution

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

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rat IgG H&L (HRP) (ab205720) at 1/50000 dilution

Observed band size: 25-37 kDa why is the actual band size different from the predicted?


Exposure time: 10 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded (Panel A) HEK-293T transfected with a TVA construct and (Panel B) HEK-293T transfected with empty plasmid, labeling TVA with ab271292 at 1/100 (10.98 µg/ml) dilution followed by a ready to use Goat Anti-rat IgG H&L (HRP polymer) (ab214882). Positive staining on (Panel A) HEK-293T transfected with a TVA construct, no staining on (Panel B) HEK-293T transfected with empty plasmid. The section was incubated with ab271292 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 Goat Anti-rat IgG H&L (HRP polymer) (ab214882).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T (human embryonic kidney) cells transfected with myc-tagged TVA expression vector labelling TVA with ab271292 at 1/50 (21.96 µg/ml) dilution, followed by ab150157 Goat Anti-rat IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in HEK-293T cells transfected with myc-tagged TVA expression vector. Myc-Tag mouse mAb (Alexa Fluor^® 647) was used as a counterstain at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody control: Used PBS instead of ab271292, followed by ab150157 Goat Anti-rat IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution.

 

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HEH-293T (human embryonic kidney epithelial cell) (transfected with myc tagged TVA construct) cells labelling TVA with ab271292 at 1/1000 dilution (0.1µg) (Right panel) compared with a rat monoclonal IgG isotype control (Left panel). Goat F(ab)2 Anti-Rat IgG Fc (Alexa Fluor^® 488, ab150161) was used as the secondary antibody at a 1/2000 dilution.

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling TVA with ab271292 at 1/100 (10.98 µg/ml) dilution followed by a ready to use Goat Anti-rat IgG H&L (HRP polymer) (ab214882). Negative control: No staining on human cerebrum. The section was incubated with ab271292 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 Goat Anti-rat IgG H&L (HRP polymer) (ab214882).

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 TVA with ab271292 at 1/100 (10.98 µg/ml) dilution followed by a ready to use Goat Anti-rat IgG H&L (HRP polymer) (ab214882). Negative control: No staining on mouse cerebrum. The section was incubated with ab271292 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 Goat Anti-rat IgG H&L (HRP polymer) (ab214882).

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 TVA with abab271292 at 1/100 (10.98 µg/ml) dilution followed by a ready to use Goat Anti-rat IgG H&L (HRP polymer) (ab214882). Negative control: No staining on rat cerebrum. The section was incubated with ab271292 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 Goat Anti-rat IgG H&L (HRP polymer) (ab214882).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.