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BD Transduction Laboratories™ Purified Mouse Anti-GSK-3β
Clone 7/GSK-3b (RUO)
Western blot analysis of GSK-3β on a RSV-3T3 lysate. Lane 1: 1:2500, lane 2: 1:5000, lane 3: 1:10000 dilution of the anti-GSK-3β antibody.
Immunofluorescent staining of HeLa (ATCC CCL-2) cells. Cells were seeded in a 96 well imaging plate (Cat. No. 353219) at ~10 000 cells per well. After overnight incubation, cells were stained using the alcohol perm protocol and the anti-GSK-3β antibody. The second step reagent was Alexa Fluor® 555 goat anti-mouse IgG (Invitrogen). The image was taken on a BD Pathway™ 855 Bioimager using a 20x objective. This antibody also stained A549 (ATCC CCL-185) and U-2 OS (ATCC HTB-96) and cells and worked with both the Triton™ X-100 and alcohol perm protocols (see Recommended Assay Procedure).
Western blot analysis of GSK-3β on a RSV-3T3 lysate. Lane 1: 1:2500, lane 2: 1:5000, lane 3: 1:10000 dilution of the anti-GSK-3β antibody.
Immunofluorescent staining of HeLa (ATCC CCL-2) cells. Cells were seeded in a 96 well imaging plate (Cat. No. 353219) at ~10 000 cells per well. After overnight incubation, cells were stained using the alcohol perm protocol and the anti-GSK-3β antibody. The second step reagent was Alexa Fluor® 555 goat anti-mouse IgG (Invitrogen). The image was taken on a BD Pathway™ 855 Bioimager using a 20x objective. This antibody also stained A549 (ATCC CCL-185) and U-2 OS (ATCC HTB-96) and cells and worked with both the Triton™ X-100 and alcohol perm protocols (see Recommended Assay Procedure).
Product Details
BD Transduction Laboratories™
Mouse (QC Testing), Human, Rat, Dog, Chicken (Tested in Development)
Mouse IgG1
Rat GSK-3β aa. 1-160
Western blot (Routinely Tested), Bioimaging (Tested During Development), Immunohistochemistry, Immunoprecipitation (Reported)
46 kDa
250 µg/ml
AB_397601
Aqueous buffered solution containing BSA, glycerol, and ≤0.09% sodium azide.
RUO
Preparation And Storage
Store undiluted at -20°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
Recommended Assay Procedures
Bioimaging
1. Seed the cells in appropriate culture medium at ~10,000 cells per well in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219) and culture overnight.
2. Remove the culture medium from the wells, and fix the cells by adding 100 μl of BD Cytofix™ Fixation Buffer (Cat. No. 554655) to each well. Incubate for 10 minutes at room temperature (RT).
3. Remove the fixative from the wells, and permeabilize the cells using either BD Perm Buffer III, 90% methanol, or Triton™ X-100:
a. Add 100 μl of -20°C 90% methanol or Perm Buffer III (Cat. No. 558050) to each well and incubate for 5 minutes at RT.
OR
b. Add 100 μl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.
4. Remove the permeabilization buffer, and wash the wells twice with 100 μl of 1× PBS.
5. Remove the PBS, and block the cells by adding 100 μl of BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) to each well. Incubate for 30 minutes at RT.
6. Remove the blocking buffer and add 50 μl of the optimally titrated primary antibody (diluted in Stain Buffer) to each well, and incubate for 1 hour at RT.
7. Remove the primary antibody, and wash the wells three times with 100 μl of 1× PBS.
8. Remove the PBS, and add the second step reagent at its optimally titrated concentration in 50 μl to each well, and incubate in the dark for 1 hour at RT.
9. Remove the second step reagent, and wash the wells three times with 100 μl of 1× PBS.
10. Remove the PBS, and counter-stain the nuclei by adding 200 μl per well of 2 μg/ml Hoechst 33342 (e.g., Sigma-Aldrich Cat. No. B2261) in 1× PBS to each well at least 15 minutes before imaging.
11. View and analyze the cells on an appropriate imaging instrument.
Bioimaging: For more detailed information please refer to http://www.bdbiosciences.com/support/resources/protocols/ceritifed_reagents.jsp
Western blot: For more detailed information please refer to http://www.bdbiosciences.com/pharmingen/protocols/Western_Blotting.shtml
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Triton is a trademark of the Dow Chemical Company.
Companion Products
Stain Buffer (FBS) RUO
Size
500 ML
Cat No.
554656
Fixation Buffer RUO
Size
100 ML
Cat No.
554655
Perm Buffer III RUO
Size
125 ML
Cat No.
558050
Cat No.
353219
HRP Goat Anti-Mouse Ig RUO
Size
1 ML
Cat No.
554002
610202 Rev. 2
Antibody Details
7/GSK-3b
Glycogen Synthase Kinase-3β (GSK-3β) is a serine/threonine kinase that affects glycogen metabolism by phosphorylating and, thus, down-regulating the activity of muscle glycogen synthase. GSK-3β is identical to the Tau Protein Kinase 1 (TPK 1) which plays a role in the formation of the histopathological lesions of Alzheimer's disease (AD). In AD, many neurons contain intracytoplasmic neurofibrillary tangles (NFT) that are composed of ubiquitin and highly phosphorylated tau proteins, members of a family of microtubule-assocaited phosphoproteins. Phosphorylation of the tau proteins by GSK-3β converts them into paired helical filaments (PHF) which are found in NFT and degenerative neurites of senile plaques. This ability of GSK-3β to convert tau proteins to PHF has been demonstrated in vitro using primary cultures of embryonic rat hippocampal neurons.
610202 Rev. 2
Format Details
Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
610202 Rev.2
Citations & References
View product citations for antibody "610202" on CiteAb
Development References (5)
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Bijur GN, Jope RS. Proapoptotic stimuli induce nuclear accumulation of glycogen synthase kinase-3 beta. J Biol Chem. 2001; 276(40):37436-37442. (Clone-specific: Flow cytometry, Immunofluorescence, Immunoprecipitation, Western blot). View Reference
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Etienne-Manneville S, Hall A. Cdc42 regulates GSK-3beta and adenomatous polyposis coli to control cell polarity. Nature. 2003; 421(6924):753-756. (Clone-specific: Immunofluorescence, Immunoprecipitation, Western blot). View Reference
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Lucas JJ, Hernandez F, Gomez-Ramos P, Moran MA, Hen R, Avila J. Decreased nuclear beta-catenin, tau hyperphosphorylation and neurodegeneration in GSK-3beta conditional transgenic mice. EMBO J. 2001; 20(1-2):27-39. (Clone-specific: Immunohistochemistry). View Reference
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Morisco C, Seta K, Hardt SE, Lee Y, Vatner SF, Sadoshima J. Glycogen synthase kinase 3beta regulates GATA4 in cardiac myocytes. J Biol Chem. 2001; 276(30):28586-28597. (Clone-specific: Gel shift, Immunofluorescence, Immunoprecipitation, Western blot). View Reference
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Takashima A, Noguchi K, Sato K, Hoshino T, Imahori K. Tau protein kinase I is essential for amyloid beta-protein-induced neurotoxicity. Proc Natl Acad Sci U S A. 1993; 90(16):7789-7793. (Biology). View Reference
610202 Rev. 2
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.