Immunohistochemical staining of paraffin embedded human cervical carcinoma with purified ab133497 at a working dilution of 1/100. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunofluorescence staining of Jurkat cells with purified ab133497 at a working dilution of 1/500, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab133497 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

All lanes : Anti-Flotillin 1 antibody [EPR6041] (ab133497) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : FLOT1 knockout HEK293T cell lysate
Lane 3 : HAP1 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 47 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab133497 observed at 50 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab133497 Anti-Flotillin 1 antibody [EPR6041] was shown to specifically react with Flotillin 1 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab267276 (knockout cell lysate ab257109) was used. Wild-type and Flotillin 1 knockout samples were subjected to SDS-PAGE. ab133497 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

ab133497 (purified) at 1/60 immunoprecipitating Flotillin 1 in 10 μg K562 (Lanes 1 and 2, observed at 48 kDa). Lane 3 - PBS. For western blotting, a HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG was used as the secondary antibody (1/1500). Blocking buffer and concentration: 5% NFDM/TBST Dilution buffer and concentration: 5% NFDM/TBST

Flow Cytometry analysis of Jurkat (human acute T cell leukemia) cells labeling Flotillin 1 with purified ab133497 at 1/50 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^®488)(1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.

Immunohistochemical analysis of paraffin embedded Human colon tissue labelling Flotillin 1 using unpurified ab133497 at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: Flotillin 1 knockout HAP1 whole cell lysate (20 µg)

Lanes 1 - 2: Merged signal (red and green). Green - ab133497 observed at 47 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab133497 was shown to specifically react with Flotillin 1 in wild-type HAP1 cells as signal was lost in Flotillin 1 knockout cells. Wild-type and Flotillin 1 knockout samples were subjected to SDS-PAGE. Ab133497 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/10000 dilution and 1/20000 dilution respectively. Blots were developed 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 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Flotillin 1 antibody [EPR6041] (ab133497) at 1/10000 dilution (purified)

Lane 1 : K562 cell lysate
Lane 2 : A431 cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 47 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

All lanes : Anti-Flotillin 1 antibody [EPR6041] (ab133497) at 1/10000 dilution (purified)

Lane 1 : mouse brain lysate
Lane 2 : mouse kidney lysate
Lane 3 : rat kidney lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit IgG (H+L)

Predicted band size: 47 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

All lanes : Anti-Flotillin 1 antibody [EPR6041] (ab133497) at 1/10000 dilution

Lane 1 : K562 cell lysate
Lane 2 : A431 cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : HeLa cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 47 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?