Cells were harvested in lysis buffer (Sigma) supplemented with a 1% protease inhibitor PMSF (Beyotime) and centrifuged at 13,000 rpm for 20 minutes at 4°C. Protein extracts were separated on 12% SDS-PAGE gels and immunoblotted with primary antibodies SMN (1:500, Santa Cruz) and Tubulin (1:10,000, Beyotime). HRP-conjugated secondary antibodies (1:5,000, Santa Cruz) were used to detect primary antibodies and proteins were visualized by chemiluminescence (Millipore).
Tubulin
Manufactured by Beyotime
Sourced in United States, China
Tubulin is a globular protein that is a fundamental component of microtubules, which are cytoskeletal structures found in eukaryotic cells. Tubulin plays a crucial role in various cellular processes, including cell division, cell motility, and intracellular transport.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (6 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Gapdh, by Beyotime (5 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions)
Ecl system, by Beyotime (3 mentions)
N cadherin, by Cell Signaling Technology (3 mentions)
Vimentin, by Cell Signaling Technology (3 mentions)
β actin, by Beyotime (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Bca kit, by Beyotime (2 mentions)
Actin, by Beyotime (2 mentions)
Cyclin a2, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated secondary antibody hrp conjugated secondary antibodies goat anti mouse igg and goat anti rabbit igg, by Beyotime (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Phenylmethanesulfonyl fluoride, by Beyotime (2 mentions)
α sma, by Abcam (2 mentions)
Phosopho erk1 2 thr202 tyr204, by Cell Signaling Technology (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Erk1 2, by Cell Signaling Technology (2 mentions)
Collagen 1, by Abcam (2 mentions)
38 protocols using tubulin
1
SDS-PAGE Protein Extraction and Detection
2
VEGF Expression Analysis in ESCC Cells
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RIPA lysis buffer (Thermo Scientific, USA) was utilized for protein extraction from ESCC cells after transfection for 48 h, followed by protein quantification using a BCA kit (Beyotime Biotechnology, Nantong, China). Afterward, 20 µg of protein sample was separated by electrophoresis, transferred to the PVDF membrane, and blocked in the blocking solution at 37°C on a shaker for 1 h. Afterward, membranes were reacted with primary antibody at 4°C overnight, reacted with proper secondary antibody at 37°C for 1 h, visualized by chemiluminescence (ECL) (YEASEN, Shanghai, China), and processed and analyzed by a gel imager. Tubulin was used as the internal control in the experiment. The antibodies used in Western blot were as follows: VEGF (1 : 1000, YEASEN), Tubulin (1 : 1000, Beyotime Biotechnology), and HRP-conjugated secondary antibody (1 : 1000, Beyotime Biotechnology). The assay was performed in triplicate.
3
Investigating LC3 and GAPDH Protein Dynamics
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LC3 (catalog number: 3868S) and GAPDH (catalog number: 5174S) antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). Cell Counting Kit-8 was purchased from Beyotime (Shanghai, China). Actin (catalog number: C2203S) and tubulin (catalog number: C1051S) staining reagents were purchased from Beyotime (Shanghai, China). Matrix (catalog number: 356234) was purchased from Corning Incorporated (New York, NY, China). The sequencing and packaging of the virus carrying LC3B (catalog number: NM_022818) was completed by Genechem Co., Ltd. (Shanghai, China). Triptonodiol (B32757) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China).
4
Protein Expression Analysis of PM Muscle
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RIPA lysis buffer (Beyotime Biotechnology, Jiangsu, China), which containing phosphatase and protease inhibitors, was added into frozen PM muscle samples and then homogenized. The homogenate was centrifuged at 4°C for 20 min at 12,000 g, and the supernatant was collected. The BCA kit was used to detect the protein concentration. Equal amounts of total protein were electrophoresed on SDS-PAGE (6% or 10%) and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA). At room temperature, bovine serum albumin (5%) was used to block the membranes for 1.5 h. Next, the membranes were incubated in primary antibodies against PMCA1, NCX1, TRPC1 (Zen-Bioscience, Chengdu, China), MCU (Abclonal, Wuhan, China), SERCA1 (Abcam, Cambridge, UK), IP3R (ProteinTech, Wuhan, China), and Tubulin (Beyotime, Shanghai, China) overnight at 4°C with gentle shaking. Finally, at room temperature, the membranes were incubated by using the corresponding secondary antibodies (Cell Signaling Technology, Danvers, MA) for 1 h. The protein bands were treated with an ECL chemiluminescence detection kit (Vazyme Biotechnology Co., Ltd.) and then visualized with the ChemiDoc TM Imaging System (BIO-RAD, Singapore). Quantity One software (Bio-Rad) was used to quantify the density of bands and the relative protein expression was calculated by ImageJ software with Tubulin as a normalized control.
5
Western Blotting Protocol for OS-9, HIF-1α, and Cytoskeletal Proteins
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Western blotting were conducted under standard procedures (Cai et al, 2010 ). Briefly, cells were broken open to obtain proteins using RIPA. Proteins were separated by 8% SDS–PAGE and then transferred to PVDF membrane (Bio-Rad, Hercules, CA, USA). After blocking in 5% nonfat milk, the membranes were incubated with the following primary antibodies: OS-9 (1 : 1000; Novus Biologicals, Littleton, CO, USA), HIF-1α (1 : 1000; BD Biosciences), GAPDH (1 : 10000; KangChen, Shanghai, China) and tubulin (1 : 1000; Beyotime, Beijing, China), and then the secondary antibodies: rabbit anti-mouse IgG and goat anti-rabbit IgG (1 : 10000; SAB, Bethesda, MD, USA). All antibodies were diluted in nonfat dry milk. The immunoreactive protein bands were visualised by ECL Kit (Pierce, Thermo Fisher Scientific, IL, USA). The experiment was performed three separate times.
6
Western Blot Analysis of Cell Signaling Proteins
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RIPA lysis buffer (Beyotime, Shanghai, China) containing Phenylmethanesulfonyl fluoride (Beyotime) was used to lyse cells. After lysis, cell lysates were denatured for 30 min at 100 °C. Then, proteins were separated with 8% and 10% SDS-PAGE gel. Separated proteins were transferred to polyvinylidene difluoride membranes. The membranes blocked in 5% BSA (bovine serum albumin) at room temperature for 2 h. The primary antibodies against CDK1, Cyclin A2, Cyclin B1, MMP-7, MMP-2, Snail, Slug, p-MEK, P-ERK, AKT, p-AKT, and MET, which were purchased from Cell Signaling Technology (Proteintech, Chicago, IL, USA) as well as Tubulin purchased from Beyotime, incubated with membranes overnight at 4 °C. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (HRP-conjugated secondary antibodies (goat anti-mouse IgG and goat anti-rabbit IgG, 1:10000, Beyotime, China) at room temperature for 2 h. Finally, ECL system (Beyotime, China) and ProXima chemiluminescence gel imaging system (Isogen, De Meern, Utrecht, The Netherlands) were used to visualize and captured proteins.
7
Co-Immunoprecipitation of ZHX2 Protein
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Cell lysate was prepared using RIPA (Beyotime) or Pierce IP lysis buffer (Thermo fisher scientific) containing protease inhibitor cocktail. Protein quantification was performed by BCA assay (Beyotime). Membrane was blocked in 5% skin‐milk for 1 hour and then incubated with primary antibody overnight at 4C. Horseradish peroxidase‐conjugated secondary antibody (Beyotime) was then added for 1 hour at room temperature. Briefly, protein supernatant for Co‐IP was incubated with Protein A/G PLUS‐Agarose (Santa Cruz Biotechnology) beads for 3 hour at 4°C. The supernatant was transferred to a new tube, added anti‐ZHX2 or anti‐IgG antibodies, and incubated for 1 hour at 4°C. The mixture was incubated with beads under rotary agitation overnight at 4°C, then centrifuged and discarded the supernatant. Beads were collected and washed for three times. The 2 × loading‐buffer was added to beads and the mixture was heated at 100℃ for 10 minutes. Protein‐antibody complexes were determined by immunoblot. The following primary antibodies were used: ZHX2(GeneTex), NF‐κB (CST), Tubulin (Beyotime), and IgG (ABclonal).
8
Protein Extraction and Western Blot Analysis
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Protein extraction and western blotting were performed as previously reported.56 (link) Briefly, total proteins were extracted from lysis buffer containing protease inhibitor cocktail (Kangchen, cat. no. KC-415), and nuclear or cytoplasmic proteins were extracted using the Minute Cytoplasmic and Nuclear Fractionation kit (Invent, cat. no. SC-003). Proteins (30 μg) were subjected to SDS-PAGE using polyacrylamide gels. Immunoblotting was performed using specific antibodies (ANP, Beyotime, cat. no. AF7608; BNP, Abcam, cat. no. ab236101; β-MHC, Abcam, cat. no. ab172967; NF-κB p65, Cell Signaling, cat. no. D14E12; IκBα, Cell Signaling, cat. no. 9242; G3BP2, Affinity Biosciences, cat. no. DF4387, TUBULIN, Beyotime, cat. no.AF1216; GAPDH, Beyotime, cat. no. AF1186 and Lamin B1, Proteintech, cat. no.12987-1-AP) to evaluate the expression of proteins. The image was acquired using the Odyssey infrared fluorescence imaging system (Li-Cor, Lincoln, NE). Band intensity was quantified by a densitometric analysis using ImageJ software (National Institutes of Health).
9
Western Blot Analysis of Cellular Proteins
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Western blot was performed as the previously described [26 (link)]. Briefly, total protein was extracted using RIPA buffer added with PMSF and phosphatase inhibitors. Protein concentration was determined using BCA protein assay (Millipore, Switzerland). Then, 20 μg of protein was separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (PVDF, Millipore, USA). After blocked with 5% non-fat milk, the membrane was incubated with primary antibody against PCNA (1:2000), Snail (1:1000), Vimentin (1:1000), E-cadherin (1:1000), N-cadherin (1:1000), integrin β1 (1:1000), Shc (1:1000), phosopho-Shc (1:2000), ERK1/2 (1:1000) or phosopho-ERK1/2 (Thr202/Tyr204) (1:2000) (Cell Signaling Technology, USA), periostin (1:1000), collagen I (1:5000), α-SMA (1:300) or anti-vitamin D receptor antibody (1:1000) (Abcam, USA), GAPDH (1:1000), tubulin (1:1000) or β-actin (1:1000) (Beyotime, China) at 4 °C overnight and the corresponding HRP-conjugated secondary antibody for 1 h at room temperature on the next day. The membrane was developed with enhanced chemiluminescence (ECL; New Cell & Molecular Biotech Co., China).
10
Comprehensive Western Blot Analysis of Cellular Markers
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Western blot was carried out as previously described [26 (link)]. Total proteins were extracted with lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) premixed with phenylmethanesulfonyl fluoride (PMSF) and phosphatase inhibitor (Roche). After samples were loaded into gels, electrophoresis, transferring and immunostaining were conducted. The primary antibodies used were: PCNA (1:2000), vimentin (1:1000), E-cadherin (1:1000), N-cadherin (1:1000), Nanog (1:1000), ERK1/2 (1:1000), phosopho-ERK1/2 (Thr202/Tyr204) (1:2000) (Cell Signaling Technology, USA), Tubulin (1:1000) and GAPDH (1:1000) (Beyotime, China). The immunoreactive bands were detected by enhanced chemiluminescence (New cell & Molecular Biotech, China).
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