Cells were scraped into ice-cold phosphate buffered saline (PBS) containing 1x Sigma Fast Protease Inhibitor Cocktail (Roche) and collected as a pellet by centrifugation (500xg, 5 min, 4°C). Cells were lysed (45 min., 4°C) by agitation in modified RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2% NP-40, 0.25% Deoxycholate, 0.1% SDS, 1mM NaF, 1mM DTT, 1X SigmaFast, 10% glycerol), followed by centrifugation at 20,000xg for 30 min. at 4°C and determination of the total protein concentration in each lysate using the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). Equal amounts of each lysate (50 μg) were fractionated by 8% PAGE, transferred to nitrocellulose membrane and blocked with 0.1% Casein in 0.2x PBS for 1 hour at room temperature. Membranes were incubated with protein-specific antibodies overnight at 4°C followed by 1 hour treatment with secondary antibodies prior to quantification using chemiluminescence or LiCOR Odyssey. Antibodies used are as follows: anti-LDAH (described previously14 (link)); anti-GFP (NB600–308) from Novus Biologicals, Centennials, CO; anti-Calnexin (ADI-SPA-865-D) from Enzo Life Sciences, Farmingdale, NY; anti-ATGL (2138S) from Cell Signaling Technology, Danvers, MA; anti-αTubulin (T6199) from Sigma-Aldrich, St. Louis, MO.
Anti gfp nb600 308
Manufactured by Novus Biologicals
Sourced in United States
The Anti-GFP (NB600-308) is a primary antibody that specifically binds to the Green Fluorescent Protein (GFP). It can be used to detect and localize GFP-tagged proteins in various applications such as Western blotting, immunocytochemistry, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Merck Group (3 mentions)
Sigma fast protease inhibitor cocktail, by Roche (2 mentions)
Anti ldah, by Novus Biologicals (2 mentions)
Anti calnexin adi spa 865 d, by Enzo Life Sciences (2 mentions)
Anti atgl 2138s, by Cell Signaling Technology (2 mentions)
Anti α tubulin t6199, by Merck Group (2 mentions)
Iblot, by Thermo Fisher Scientific (2 mentions)
Anti ube3a, by BD (2 mentions)
Anti β tubulin, by Merck Group (2 mentions)
Bis tris gel, by Thermo Fisher Scientific (2 mentions)
T5168, by Merck Group (2 mentions)
Anti α tubulin, by Merck Group (2 mentions)
Irdye 800cw goat anti mouse, by LI COR (2 mentions)
Odyssey system, by LI COR (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Enhanced chemiluminescence kit, by Merck Group (1 mentions)
Anti gapdh lf p a0212, by AbFrontier (1 mentions)
Anti flag f1804, by Merck Group (1 mentions)
6 protocols using anti gfp nb600 308
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of Neuronal Proteins
3
Protein Expression Analysis Using SDS-PAGE
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The cells were lysed with RIPA buffer (Millipore, Billerica, MA, USA) containing protease inhibitor cocktail (Roche), and the lysates were quantified with a protein assay kit (Bio-Rad, Hercules, CA, USA). The cell lysates were separated using SDS-PAGE and then transferred to PVDF membranes. The proteins were identified while using appropriate antibodies. Anti-VGLL1 (10124-2-AP) was purchased from Proteintech (Rosemont, IL, USA). Anti-TEAD4 (ab58310) and anti-MMP9 (ab76003) were purchased from Abcam (Cambrige, MA, USA). Anti-GFP (NB600-308) was purchased from Novus Biologicals (Centennial, CO, USA). Anti-Myc (sc-789) and anti-HA (sc-805) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-GAPDH (LF-PA0212) was purchased from AbFrontier (Seoul, Korea). Anti-β-tubulin (2128), anti-pSer473-AKT (9271), anti-AKT (9272), anti-p-β-catenin (9561), and anti-β-catenin (9562) were purchased from Cell Signaling (Danvers, MA, USA). Anti-Flag (F1804) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The protein signal was detected while using an enhanced chemiluminescence kit (Millipore, Burlington, MA, USA).
4
Protein Extraction and Western Blot Analysis
5
Western Blot Analysis of Neuronal Proteins
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Cultured neurons and mouse tissue were homogenized and lysed in RIPA buffer (Sigma-Aldrich) containing EDTA-free cOmplete Protease Inhibitor Cocktail (Roche). Protein concentration of the supernatant was determined by the DC protein assay (Bio-Rad). 10-40 μg protein lysate was separated on a precast 4-20% Bis-Tris gel (Life Technologies) and transferred by iBlot (Life Technologies). The following primary antibodies were diluted in Odyssey blocking buffer: anti-UBE3A (611416, BD Biosciences, 1:500), anti-GFP (NB600-308, Novus Biologicals, 1:500), anti-β-Tubulin (T9026, Sigma, 1:20,000), and anti-α-Tubulin (T5168, Sigma, 1:8,000). Following primary antibody incubation, membranes were probed with goat anti-rabbit IRDye 680LT (LiCor) or goat anti-mouse IRDye 800CW (LiCor) and imaged and quantified using the LiCor Odyssey system.
6
O-GlcNAc Signaling in Amino Acid Starvation
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