Recombinant Anti-GSK3 beta + GSK3 alpha antibody [EPR18814-102] - BSA and Azide free (ab226169)

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

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

Lysates/proteins at 20 µg per lane.

Predicted band size: 47, 52 kDa

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185141).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185141).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185141).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185141).

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. 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185141). 

 

 

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185141).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185141).

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