All lanes : Anti-Annexin-6/ANXA6 antibody [EPR17308] - N-terminal (ab199422) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ANXA6 knockout HeLa cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : Raji cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 76 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab199422 observed at 75 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab199422 was shown to react with Annexin-6/ANXA6 in HeLa wild-type cells in western blot with loss of signal observed in ANXA6 knockout cell line ab265677 (ANXA6 knockout cell lysate ab257351). Wild-type and ANXA6 knockout HeLa cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab199422 and ab8245 (Mouse anti-GAPDH antibody [6C5]) 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 hour at room temperature before imaging.

Immunofluorescent analysis of 100% methanol-fixed, 0.1% Triton X-100 permeabilized K562 (Human chronic myelogenous leukemia cells from bone marrow) cells labeling Annexin-6/ANXA6 with ab199422 at 1/100, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 (green). Cytoplasm staining on K562 cell line is observed (Subcellular Location: Cytoplasm [UniProt]). The nuclear counterstain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 (red).

The negative controls are as follows:-
-ve control 1 - ab199422 at 1/1000 followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500.
-ve control 2. - ab7291 (anti-Tubulin mouse mAb) at 1/1000 followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500.

All lanes : Anti-Annexin-6/ANXA6 antibody [EPR17308] - N-terminal (ab199422) at 1/10000 dilution

Lane 1 : Raji (Human Burkitt's lymphoma) whole cell lysate
Lane 2 : HeLa (Human epithelial cells from cervix adenocarcinoma ) whole cell lysate
Lane 3 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Developed using the ECL technique.

Predicted band size: 76 kDa
Observed band size: 68 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

Blocking and diluting buffer was 5% NFDM/TBST.

The observed MW is consistent with what has been described in the literature (PMID:12140262).

All lanes : Anti-Annexin-6/ANXA6 antibody [EPR17308] - N-terminal (ab199422) at 1/1000 dilution

Lane 1 : Mouse brain tissue lysate
Lane 2 : Mouse kidney tissue lysate
Lane 3 : Mouse spleen tissue lysate
Lane 4 : Rat brain tissue lysate
Lane 5 : Rat heart tissue lysate
Lane 6 : Rat kidney tissue lysate
Lane 7 : Rat spleen tissue lysate
Lane 8 : PC-12 (Rat adrenal gland pheochromocytoma) cell lysate
Lane 9 : NIH/3T3 (Mouse embryo fibroblast) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Developed using the ECL technique.

Predicted band size: 76 kDa
Observed band size: 68 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

Blocking and diluting buffer was 5% NFDM/TBST.
The observed MW is consistent with what has been described in the literature (PMID:12140262).

Immunofluorescent analysis of 100% methanol-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Annexin-6/ANXA6 with ab199422 at 1/100, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 (green). Cytoplasm staining on HeLa cell line is observed (Subcellular Location: Cytoplasm [UniProt]). The nuclear counterstain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 (red).
The negative controls are as follows:-
-ve control 1 - ab199422 at 1/1000 followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500.
-ve control 2. - ab7291 (anti-Tubulin mouse mAb) at 1/1000 followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500.

Annexin-6/ANXA6 was immunoprecipitated from 1mg of Raji (Human Burkitt's lymphoma cell line) whole cell extract with ab199422 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab199422 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1: Raji whole cell extract 10 µg (Input).

Lane 2: ab199422 IP in Raji whole cell extract.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab199422 in Raji whole cell extract.

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