To confirm the 2-DE results, Western blot analysis was conducted using the livers of 3-week-old offspring from the AdLib/AdLib (n = 4 males, n = 4 females), AdLib/FR, FR/AdLib, and FR/FR groups (n = 5 males, n = 5 females/group). Briefly, equal amounts of lysate (40 μg) were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Amersham Biosciences, Piscataway, NJ) at 80 V for 2 h. The membrane was blocked for 1 h in 2.5% skim milk in Tris-buffered saline (TBS) with 0.01% Tween-20 (TBS-T). Next, the membrane was washed in TBS-T and incubated with antimethylenetetrahydrofolate dehydrogenase 1 (1:1000; GeneTex Inc., Irvine, CA), antibetaine-homocysteine-S-methyltransferase (1:1000; Santa Cruz Biotechnology, Dallas, TX), anti-ATP synthase subunit beta (1:2000; GeneTex Inc.), and anti-αtubulin (1:2000; AbFrontier, Seoul, Korea) antibodies overnight at 4 °C. The membrane was washed in TBS-T. Secondary anti-rabbit or anti-mouse HRP-conjugate (Santa Cruz Biotechnology) was then added at 1:3000 dilution in blocking buffer for 1 h at room temperature. The membranes were then washed again in TBS-T, and developed using enhanced chemiluminescence reagents (Santa Cruz Biotechnology).
Anti-α-tubulin
Manufactured by AbFrontier
Sourced in United States
Anti-α-tubulin is a primary antibody that specifically binds to the α-tubulin subunit of the cytoskeletal protein microtubule. It can be used for the detection and visualization of microtubules in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membranes, GE Healthcare (1 mentions)
Enhanced chemiluminescence reagent, Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence detection kit, GE Healthcare (1 mentions)
Anti-V5, Thermo Fisher Scientific (1 mentions)
Anti-p21 Waf1/Cip1, Cell Signaling Technology (1 mentions)
Anti-cyclin B1, Cell Signaling Technology (1 mentions)
Anti-CDC25C, Cell Signaling Technology (1 mentions)
Anti-γH2AX, Merck Group (1 mentions)
Anti-p53, Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence detection reagents, Thermo Fisher Scientific (1 mentions)
Anti-β-actin, AbFrontier (1 mentions)
Complete Mini, Roche (1 mentions)
PVDF membranes, Merck Group (1 mentions)
Anti-HA, Cell Signaling Technology (1 mentions)
Anti-Lamin B1, Santa Cruz Biotechnology (1 mentions)
Anti-Gal4, Santa Cruz Biotechnology (1 mentions)
Anti-ERRγ, R&D Systems (1 mentions)
Anti-Myc, Cell Signaling Technology (1 mentions)
Anti-Flag, Cell Signaling Technology (1 mentions)
Anti-GFP, Santa Cruz Biotechnology (1 mentions)
7 protocols using Anti-α-tubulin
1
Western Blot Analysis of Liver Proteins
2
Immunoblot Analysis of TNNC1-V5 Expressing Cells
TNNC1-V5-expressing cells were harvested and lysed using RIPA buffer as described previously (Jung et al., 2015a (link)). Primary antibodies used in immunoblot analyses are as follows: anti-V5 (Invitrogen), anti-p53 (Santa Cruz Biotechnology, USA), anti-p21 Waf1/Cip1 (Cell Signaling Technology, USA), anti-CDC25C (Cell Signaling Technology), anti-Cyclin B1 (Cell Signaling Technology), anti-α-tubulin (AbFrontier, Korea), and anti-γH2AX (Millipore, USA). Peroxidase-conjugated secondary antibodies were used for detection in combination with AbSignal Western Blotting Detection Reagent kit (Abclon, Korea) or enhanced chemiluminescence detection kit (Amersham-Pharmacia Biotech, USA) following the manufacturer’s protocols.
3
Analysis of ER-α and HER2 in MCF-7 Cells
MCF-7 cells were cultured overnight at a density of 3 × 105 cells/well. The cells were then treated either with a CHE or with vehicle. Next the samples were homogenized using 400 μL lysing buffer containing 150 mmol/L KCl, 10 mM Tris, pH 7.4, 1% Triton X-100, and protease inhibitor cocktail (Complete Mini, Roche, Mannheim, Germany). The protein concentration of each cell homogenates was determined as described previously [21 (link)]. Samples consisting of 30–50 μg of protein were separated on 10% SDS-polyacrylamide gels by electrophoresis and thereafter transferred to a PVDF membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% bovine serum albumin and probed with specific primary antibodies, namely, anti-ERα (Stressgen Biotechnologies Inc., Victoria, BC, Canada), anti-pHER2/anti-tHER2 (IPVH00010, Millipore, Bedford, MA, USA), anti-α-tubulin (AbFrontier, Seoul, Korea), and anti-β-actin (AbFrontier, Seoul, Korea). The immunoreactive bands were visualized using enhanced chemiluminescence detection reagents (Thermo Scientific, Bremen, Germany) and quantified by Multigauge software analysis (Fuji Photo Film Co., Ltd., Tokyo, Japan).
4
Immunoblot Analysis of Protein Abundance
Whole-cell extracts were prepared with lysis buffer (50 mM Tris-pH7.5, 150 mM NaCl, 1% NP-40, and 5 mM EDTA). Equal amounts of total proteins were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were immunoblotted with anti-FLAG, anti-HA, anti-Myc (Cell Signaling Technology, Danvers, MA, USA), anti-GFP, anti-Gal4, anti-Lamin B1 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-α-tubulin (AbFrontier, Seoul, Republic of Korea), and anti-ERRγ (R&D Systems, Minneapolis, MN, USA) antibodies. Densitometry was performed using ImageJ software (NIH, Bethesda, AR, USA).
5
Western Blot Analysis of Cellular Proteins
Cells were harvested and lysed as previously described [30 (link)]. Whole cell lysates were separated using SDS-PAGE and transferred to a PVDF membrane. Membrane was blocked with 5% skim milk for 1h and incubated with specific antibodies at 4 °C overnight with rotation. The antibodies were obtained from the following sources: anti-poly (ADP-ribose) polymerase (PARP) and anti-Akt, anti-phospho-Akt (Ser 473) were from Cell Signaling (Danvers, MA, USA); anti-GST and anti-GFP antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-His and anti-HA were from ABM (Richmond, BC, Canada); anti-α-tubulin and anti-Trx1 were from AB Frontier (Seoul, Republic of Korea); anti-SELENOW was from Origene (Rockville, MD, USA). After incubating with HRP conjugated secondary antibody for 1 h, immunoreactive bands were visualized using a West Pico Enhanced ECL Detection kit (Pierce, Rockford, IL, USA).
6
Western Blot Analysis of Cellular Proteins
Cells were harvested, washed twice with cold PBS, and lysed using a lysis buffer consisting of 25 mM Tris HCl (pH 8.0), 1 mM EDTA, 150 mM NaCl, and 0.5% NP40. After a 10-min incubation on ice, the cell lysate was centrifuged at 13,000 rpm at 4°C for 10 min. Equal amounts of protein from extracts were separated using SDS-PAGE and transferred to nitrocellulose membranes. The blots were blocked with 5% skim milk and incubated with primary antibodies, which were diluted in the range of 1:1,000 to 1:5,000. After washing with TBS-T (5 mM Tris HCl [pH 7.4], 150 mM NaCl, and 0.01% Tween 20), secondary antibodies were added at a dilution of 1:10,000 for a 1-h incubation at room temperature. Protein signals were detected using a western blot detection kit (AbFrontier, Korea). The following primary antibodies were used in this study: anti-β-actin (Merck Millipore, USA), anti-α-tubulin (AbFrontier), anti-menin (Bethyl Laboratories, USA), anti-cleaved-PARP (Cell Signaling Technology, USA), anti-AR (Santa Cruz Biotechnology, USA), and anti-Lamin B1 (Thermo Fisher Scientific).
7
Antibody Characterization for Histone Regulation
The following antibodies were used in this study: anti-HIRA (Active Motif, Carlsbad, CA, USA, WC119 and WC15), anti-Asf1a (Cell Signaling, Danvers, MA, USA, 2990), anti-Cabin1, anti-UBN1 (Abcam, Cambridge, MA, USA), anti-H3 (Abcam, ab1791), anti-H4 (Millipore, Billerica, MA, USA, 07–108), anti- H4S47p (Abcam), anti-H3.3 (Millipore, 09–838), anti-H3.1 and anti-H3.3 (provided by Dr Y Ohkawa), anti-pan-Akt, anti-Akt1, anti-Akt2, anti-p-Akt (S473) (Cell Signaling, 9271), anti-phospho-serine (Abcam, ab9332), anti-phospho-threonine (Abcam, ab9337), anti-myogenin (Santa Cruz, Santa Cruz, CA, USA, sc-576), anti-MHC (Upstate, Billerica, MA, USA, 05–716), 8WG16 (Covance, Princeton, NJ, USA, MMS-126R), anti-HA (Roche, Basel, Switzerland, 11 666 606 001), anti-Flag (Sigma-Aldrich, F3165), anti-Myc (Roche, 11 667 203 001), anti-β-catenin (Santa Cruz, sc-7963), and anti-α-tubulin (AbFrontier, Seoul, Korea, LF-PA0146). The anti-p-HIRA (Abmart, Shanghai, China) antibody was generated using the phospho-peptides RPRKD(pS)RLMPV and was affinity-purified from rabbit serum using a specific column. Antibody specificity was confirmed by checking the absence of cross-reactivity with the control peptide, RPRKDSRLMPV.
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