Recombinant Anti-GSK3 beta + GSK3 alpha antibody [EPR18814-102] (ab185141)

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: GSK3 alpha knockout HAP1 whole cell lysate (20 µg)
Lane 3: GSK3 beta whole cell lysate (20 µg)
Lane 4: HeLa whole cell lysate (20 µg)
Lane 5: Hek293 whole cell lysate (20 µg)

Lanes 1 - 5: Merged signal (red and green). Green - ab185141 observed at 47/52 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab185141 was shown to specifically react with GSK3 alpha and GSK3 beta in wild-type HAP1 cells as signal was lost in GSK3 alpha and GSK3 beta knockout cells. Wild-type and GSK3 alpha and GSK3 beta knockout samples were subjected to SDS-PAGE. ab185141 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/5000 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 1/10000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling GSK3 beta + GSK3 alpha with ab185141 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on mouse testis (PMID: 22792253). Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

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

All lanes : Anti-GSK3 beta + GSK3 alpha antibody [EPR18814-102] (ab185141) at 1/1000 dilution

Lane 1 : His-tagged mouse GSK3 beta (aa218-420) recombinant protein
Lane 2 : His-tagged mouse GSK3 alpha (aa330-490) recombinant protein

Lysates/proteins at 0.01 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 47, 52 kDa


Exposure time: 1 second

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-GSK3 beta + GSK3 alpha antibody [EPR18814-102] (ab185141) at 1/5000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : Human fetal heart lysate
Lane 3 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/4000 dilution

Developed using the ECL technique.

Predicted band size: 47, 52 kDa
Observed band size: 47,52 kDa why is the actual band size different from the predicted?

 

Exposure time : Lane 1: 30 seconds; Lanes 2 and 3: 3 minutes.

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-GSK3 beta + GSK3 alpha antibody [EPR18814-102] (ab185141) at 1/5000 dilution

Lane 1 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate
Lane 2 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : A549 (human lung carcinoma cell line) whole cell lysate
Lane 4 : RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 5 : PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 6 : Mouse heart lysate
Lane 7 : Rat heart lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 47, 52 kDa
Observed band size: 47,52 kDa why is the actual band size different from the predicted?

 

Exposure time : Lanes 1 and 2:10 seconds; Lane 3: 1 minute; Lanes 4 and 5: 5 seconds; Lane 6:3 seconds; Lane 7:10 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded rat testis tissue labeling GSK3 beta + GSK3 alpha with ab185141 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on rat testis (PMID: 22792253). Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

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 HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling GSK3 beta + GSK3 alpha with ab185141 at 1/150 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HeLa cells.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryo fibroblast cell line) cells labeling GSK3 beta + GSK3 alpha with ab185141 at 1/150 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on NIH/3T3 cells.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed,90% methanol-permeabilized NIH/3T3 (mouse embryo fibroblast cell line) cell line labeling GSK3 beta + GSK3 alpha with ab185141 at 1/60 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody. 

 

 

GSK3 beta + GSK3 alpha was immunoprecipitated from 0.35 mg of NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate with ab185141 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab185141 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: NIH/3T3 whole cell lysate 10 µg (Input). 

Lane 2: ab185141 IP in NIH/3T3 whole cell lysate. 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab185141 in NIH/3T3 whole cell lysate.

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

Exposure time: 3 seconds.