All lanes : Anti-CPT2 antibody [EPR13626] - C-terminal (ab181114) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CPT2 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 74 kDa
Observed band size: 74 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab181114 observed at 74 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab181114 was shown to react with CPT2/CPT1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265931 (knockout cell lysate ab257180) was used. Wild-type HeLa and CPT2 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab181114 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 Dilution and a 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.

Immunocytochemistry/Immunofluorescence analysis of MCF-7 (human breast carcinoma) cells labelling CPT2 with purified ab181114 at 1/100. Cells were fixed with 100% methanol and permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. The cells were co-stained with ab7291, a mouse anti-tubulin antibody (1/1000) using ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) as the secondary. Nuclei couterstained with DAPI (blue).

For negative control 1, rabbit primary antibody was used, followed by anti-mouse secondary antibody (ab150120). For negative control 2, mouse primary antibody (ab7291) was used followed by anti-rabbit secondary antibody (ab150077).  

All lanes : Anti-CPT2 antibody [EPR13626] - C-terminal (ab181114) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : CPT2 knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : MCF7 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 74 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab181114 observed at 70 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab181114 was shown to specifically react with CPT2 in wild-type HAP1 cells. No band was observed when CPT2 knockout samples were examined. Wild-type and CPT2 knockout samples were subjected to SDS-PAGE.  Ab181114 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10,000 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 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-CPT2 antibody [EPR13626] - C-terminal (ab181114) at 1/2000 dilution (purified)

Lane 1 : Human fetal kidney tissue lysate
Lane 2 : MCF-7 (human breast carcinoma) whole cell lysate
Lane 3 : Mouse heart tissue lysate
Lane 4 : Mouse kidney tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

Predicted band size: 74 kDa
Observed band size: 67 kDa why is the actual band size different from the predicted?



Blocking and diluting buffer 5% NFDM/TBST

All lanes : Anti-CPT2 antibody [EPR13626] - C-terminal (ab181114) at 1/1000 dilution (purified)

Lane 1 : Rat kidney tissue lysate
Lane 2 : Rat liver tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 74 kDa
Observed band size: 67 kDa why is the actual band size different from the predicted?



Blocking and diluting buffer 5% NFDM/TBST

All lanes : Anti-CPT2 antibody [EPR13626] - C-terminal (ab181114) at 1/10000 dilution (unpurified)

Lane 1 : Human fetal liver tissue lysate
Lane 2 : Human fetal kidney tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab136636) at 1/500 dilution

Predicted band size: 74 kDa
Observed band size: 67 kDa why is the actual band size different from the predicted?

Immunohistochemical analysis of paraffin embedded rat colon tissue section labelling CPT2 with purified ab181114 at dilution of 1/50. The secondary antibody used was HRP-conjugated Goat Anti-Rabbit IgG H&L (ab97051) at dilution of 1/500. The sample was 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. 

Immunohistochemical analysis of paraffin embedded mouse kidney tissue section labelling CPT2 with purified ab181114 at dilution of 1/50. The secondary antibody used was HRP-conjugated Goat Anti-Rabbit IgG H&L (ab97051) at dilution of 1/500. The sample was 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. 

Immunohistochemical analysis of paraffin embedded human liver carcinoma tissue section labelling CPT2 with purified ab181114 at dilution of 1/50. The secondary antibody used was Goat Anti-Rabbit IgG H&L (HRP) (ab97051), at a dilution of 1/500. The sample was 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. 

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling CPT2 with unpurified ab181114 at 1/100 dilution, followed by prediluted HRP Polymer for Rabbit IgG. Counter stained with Hematoxylin.

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling CPT2 with unpurified ab181114 at 1/100 dilution, followed by prediluted HRP Polymer for Rabbit IgG. Counter stained with Hematoxylin.

Immunofluorescent analysis of 4% paraformaldehyde-fixed MCF7 cells labeling CPT2 with unpurified ab181114 at 1/100 dilution, followed by Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody at 1/200 dilution. Counter stained with Dapi.