Recombinant Anti-SGTB/SGT2 antibody [EPR17183] (ab202419)

Anti-SGTB/SGT2 antibody [EPR17183] (ab202419) at 1/2000 dilution + HepG2 (Human liver hepatocellular carcinoma) cell lysate at 20 µg

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

Predicted band size: 33 kDa
Observed band size: 37 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression profile observed is consistent with what has been described in the literature PMID: 12878599.

Anti-SGTB/SGT2 antibody [EPR17183] (ab202419) at 1/2000 dilution + Human fetal brain lysate at 10 µg

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

Predicted band size: 33 kDa
Observed band size: 37 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression profile observed is consistent with what has been described in the literature PMID: 12878599.

All lanes : Anti-SGTB/SGT2 antibody [EPR17183] (ab202419) at 1/2000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Rat brain lysate
Lane 3 : PC-12 (Rat adrenal gland pheochromocytoma) cell lysate
Lane 4 : NIH/3T3 (Mouse embyro fibroblast cells) cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 33 kDa
Observed band size: 37 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression profile observed is consistent with what has been described in the literature PMID: 12878599.

Immunohistochemical analysis of paraffin-embedded Human cerebral cortex tissue labeling SGTB/SGT2 with ab202419 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Cytoplasmic and nuclear staining on Human cerebral cortex tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse cerebral cortex tissue labeling SGTB/SGT2 with ab202419 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Cytoplasmic and nuclear staining on mouse cerebral cortex tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Rat cerebral cortex tissue labeling SGTB/SGT2 with ab202419 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Cytoplasmic and nuclear staining on rat cerebral cortex tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized SH-SY5Y (Human neuroblastoma from bone marrow cells) cells labeling SGTB/SGT2 with ab202419 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing nuclear and weakly cytoplasmic staining on SH-SY5Y cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).

The negative controls are as follows:
-ve control 1: ab202419 at 1/100 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) cells labeling SGTB/SGT2 with ab202419 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing nuclear and cytoplasmic staining on U-87 MG cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).

The negative controls are as follows:
-ve control 1: ab202419 at 1/100 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

SGTB/SGT2 was immunoprecipitated from 1mg of Mouse brain whole cell lysate with ab202419 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab202419 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: Mouse brain whole cell lysate 10 µg (Input). Lane 2: ab202419 IP in Mouse brain whole cell lysate. Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab202419 in Mouse brain whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 1 second.