Western blot was performed as previously described [40 (link)], with the following modifications. Briefly, cells were harvested and lysed, and the cellular proteins were separated by 12% SDS-PAGE and blotted onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). After being blocked with 5% skim milk for 1 h at room temperature, the PVDF membranes were probed with one of the following primary antibodies: rabbit anti-camel IgG serum (1:2000), mouse anti-PRRSV N mAb 6D10 (1 μg/mL; produced in our laboratory), or anti-β-tubulin (1:5000; Transgene, Beijing, China). Membranes were washed three times with PBST, followed by incubation with HRP-conjugated goat anti-rabbit IgG (Jackson, USA) or HRP-conjugated goat anti-mouse IgG (Jackson, USA) at a 1:5000 dilution as the secondary antibody. The reactions were visualized using an ECL chemiluminescent detection system according to the manufacturer’s instructions (Pierce, Rockford, IL, USA).
Anti β tubulin
Manufactured by Transgene
Sourced in China
Anti-β-Tubulin is a monoclonal antibody that specifically binds to the beta-tubulin subunit of microtubules. It can be used in various laboratory applications, such as Western blotting, immunocytochemistry, and flow cytometry, to detect and quantify the presence of beta-tubulin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay reagent kit, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Anti β actin, by Transgene (3 mentions)
Ecl reagents, by Smart-Lifesciences (3 mentions)
Anti p acc, by Cell Signaling Technology (2 mentions)
Anti acc, by Cell Signaling Technology (2 mentions)
Anti hk2, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail and phosphatase inhibitor cocktail, by Apexbio (2 mentions)
Anti aldoa, by Proteintech (2 mentions)
Anti ldha, by Cell Signaling Technology (2 mentions)
Anti acly, by Cell Signaling Technology (2 mentions)
Anti p acly, by Cell Signaling Technology (2 mentions)
Anti pfkfb3, by Cell Signaling Technology (2 mentions)
Anti eno2, by Proteintech (2 mentions)
Anti hadh, by Proteintech (2 mentions)
Anti ppara, by Proteintech (2 mentions)
Anti flag, by ABclonal (2 mentions)
Prestained protein ladder 26616, by Thermo Fisher Scientific (2 mentions)
Nocodazole, by Selleck Chemicals (2 mentions)
Protease inhibitor cocktail, by Apexbio (2 mentions)
14 protocols using anti β tubulin
1
Western Blot Analysis of Viral Proteins
2
Comprehensive Cell Autophagy Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
H2DCFDA, rapamycin, pyronin Y, and buthionine sulfoximine were obtained from Sigma-Aldrich Co. (USA). Bafilomycin A1 and pepstatin A were obtained from Sangon Biotech Co. (China). D-Luciferin was obtained from Goldbio Co. (USA). Polybrene, Hoechst 33342, and propidium iodide (PI) were obtained from Yeasen Biotech Co. (China). Transfection reagents DharmaFECT, Lipofectamine 2000, and Alexa Fluor 488 and 567 secondary antibodies were obtained from Thermo Fisher Scientific Inc. (USA, 1:500). Anti-LC3 (#3868, 1:1000), anti-Beclin (#3495, 1:1000), anti-ATG5 (#12994, 1:1000), anti-Oct-4 (#2750, 1:1000), and anti-p62/SQSTM1 (#7695, 1:1000) antibodies were obtained from Cell Signaling Technology (USA). Anti-RB1CC1 (sc-22709, 1:100), anti-Ki-67 (sc-23900, 1:100), and HRP-conjugated goat anti-rabbit secondary antibodies were obtained from Santa Cruz Biotechnology (USA). Anti-vimentin, HRP-conjugated goat anti-mouse, and rabbit anti-goat secondary antibodies were obtained from Boster Co. (China). Anti-β-tubulin (1:1000) and anti-β-actin (1:1000) antibodies were obtained from Transgene Biotech Co. (China). Anti-NRF2 (WL02135, 1:2000), anti-NOTCH1 (WL01991, 1:500), and anti-Lamin B (WL01775, 1:500) antibodies were obtained from Wanleibio Co. (China).
3
Optimized Cell Transfection and Signaling Pathway Inhibition
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Polyethylenimine (PEI) (764582, Sigma-Aldrich) and jetPRIME (114-15, Polyplus) were used for transfection. Quenched fluorescent reporter RNA was purchased from General Biology. Inhibitors used in this study include the following: p38 inhibitor SB203580 (HY-10256, MCE); JNK inhibitor SP600125 (HY-12041, MCE); MEK1/2 inhibitor U0126 (HY-12031, MCE); RhoA/C inhibitor (S7719, Selleck). ZAK inhibitors 6P and HY180 are gifts from Prof. Xiaoyun Lu, Jinan University. Antibodies used in this study include the following: anti-Cre (Rabbit, 15036T, CST); anti-HA (Rat, 11867423001, Roche); anti-HA (Rabbit, H6908, Sigma-Aldrich); anti-HA (Mouse, self-made); anti-SIK3 (Rabbit, self-made); anti-β-Tubulin (Mouse, HC101, TransGen Biotech); anti-ACTB (Mouse, 60008-1-Ig, Proteintech); anti-GAPDH (Mouse, 60004-1-Ig, Proteintech); anti-NeuN (Rabbit, 26975-1-AP, Proteintech); anti-Tau (Rabbit, 10274-1-AP, Proteintech); anti-MAP2 (Rabbit, 17490-1-AP, Proteintech); anti-ZAK (Rabbit, 28761-1-AP, Proteintech); anti-PKR (Rabbit, 18244-1-AP, Proteintech); anti-p-p38 (Rabbit, 4511, CST); anti-p-JNK (Rabbit, 4370, CST); anti-p-ERK1/2 (Rabbit, ET1609-42, HUABIO); HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) (SA00001-2, Proteintech); HRP-conjugated Recombinant Rabbit Anti-Mouse IgG Kappa Light Chain (SA00001-1, Proteintech).
4
Flag-Tagged Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Flag-CTL and Flag-GDPD5 plasmids were transfected into SH-SY5Y cells with Neofect™ DNA transfection reagent. After cells were transfected for 48 h, the cells were lysed by 1% SDS lysing buffer containing Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail (Apexbio, Houston, TX, USA) for Western blot analysis. The protein concentration was determined by a BCA protein assay reagent kit (Thermo Scientific, Waltham, MA, USA). All blots were, respectively, incubated with primary anti-bodies anti-flag (1:1000, ABclonal, Wuhan, China), anti-p-ACC, anti-ACC, Anti-p-ACLY, anti-ACLY, anti-PFKFB3, anti-HK2 and anti-LDHA (1:1000, Cell Signaling Technology, MA, USA), anti-ALDOA, anti-ENO2, anti-HADH and anti-PPARA (1:1000, proteintech, Wuhan, China), as well as anti-β-Tubulin (1:5000, TRANSGEN, Beijing, China). Bands were visualized with ECL Reagents (Smart-Lifesciences, Changzhou, China).
5
Molecular Mechanisms of Autophagy Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SBI‐0206965 was from Selleck (Houston, TX, USA), and MRT68921 was from Sigma (St. Louis, MS, USA). ULK1 antibody (#8054), LC3B antibody (#3868), the cell‐cycle regulation antibody sampler kit II (#9870) and the horseradish peroxidase‐linked anti‐rabbit and anti‐mouse IgG were all from Cell Signaling Technology (Danvers, MA, USA). The anti‐β‐Tubulin, anti‐(glyceraldehyde 3‐phosphate dehydrogenase) and anti‐β‐actin Ig and TransStart FastPfu DNA Polymerase were from Beijing TransGen Biotech (Beijing, China). Nocodazole was from Selleck. Propidium iodide (PI)/RNase staining buffer was from BD Pharmingen (San Diego, CA, USA). The siRNAs were ordered from Genepharm (Shanghai, China), and primers were from Sangon Biotech (Shanghai, China). HiPerFect was from Qiagen (Dusseldorf, Germany). Protease inhibitor and phosphatase inhibitor cocktails were from Roche (Basel, Switzerland), and the polyvinylidene fluoride membrane was from Millipore (Billerica, NJ, USA). GlutaMAX was from Gibco (Carlsbad, CA, USA). The secondary fluorescently conjugated antibodies, Antifade ProLong Gold, were from Molecular Probes (Eugene, OR, USA). M‐MLV Reverse Transcriptase and TRIzol were from Invitrogen (Waltham, MA, USA). Prestained Protein Ladder (26616) and M‐PER were from Thermo Pierce (Waltham, MA, USA). CellTiter‐Glo (#G7570) was from Promega (San Luis Obispo, CA, USA).
6
Investigating Metabolic Regulators in OSRC2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The GFP-CTL and GFP-HMGCS2 plasmids were transfected into OSRC2 cells with Neofect™ DNA transfection reagent. After cells were transfected for 48 h, the cells were lysed by 1% SDS lysing buffer containing Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail (Apexbio, Houston, TX, USA) for Western blot analysis. The protein concentration was determined by a BCA protein assay reagent kit (Thermo Scientific, Waltham, MA, USA). All blots were, respectively, incubated with primary anti-bodies anti-flag (1:1000, ABclonal, Wuhan, China), anti-p-ACC, anti-ACC, Anti-p-ACLY, anti-ACLY, anti-PFKFB3, anti-HK2 and anti-LDHA (1:1000, Cell Signaling Technology, MA, USA), anti-ALDOA, anti-ENO2, anti-HADH and anti-PPARA (1:1000, proteintech, Wuhan, China), and anti-β-Tubulin (1:5000, TRANSGEN, Beijing, China). Bands were visualized with ECL Reagents (Smart-Lifesciences, Changzhou, China).
7
Western Blotting Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting analysis was performed as described previously (27 (link)). Briefly, the cells were lysed with 1% SDS lysis buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Apexbio, Houston, USA). The protein concentration was determined by using a BCA protein assay reagent kit (Thermo Scientific). The protein content (18 μg) was separated by SDS-PAGE gels and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The PVDF membranes were blocked overnight by 5% fat free milk in TBST at 4°C, followed by incubation at 4°C for 12 h with primary antibodies for anti-β-actin (1:5000) (Sigma), anti-β-Tubulin (1:5000) (TransGen Biotech), anti-ACSL1 (1:1000) (Cell Signaling Technology), ACC (1:1000) (Cell Signaling Technology), HK2 (1:1000) (Cell Signaling Technology), ACAA1 (1:1000) (Cell Signaling Technology), E-cadherin (1:1000) (Bioworld), N-cadherin (1:1000) (Bioworld), Twist1 (1:1000) (Cell Signaling Technology), respectively. The membranes were washed with TBST and incubated for 1.5 h with the corresponding HRP-conjugated secondary antibodies. The bands were visualized with ECL reagents (Merck, Billerica, MA, USA). The western blots were analyzed with the Image Lab Software (BIO-RAD, USA), and the program included an application with protein gels; the image exposure time to was set to 16 s.
8
Western Blot Immunoblotting Procedure
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For Western blots, the samples resolved by SDS-PAGE were transferred onto polyvinylidene fluoride (PVDF) membranes. Non-specific protein interactions were blocked with 5% skim milk in TBST buffer (TBS buffer containing 0.05% Tween-20) for 1 h at room temperature. The membranes were incubated in TBST buffer for 2 h at room temperature with the appropriate primary antibodies: anti-chDDX3X and anti-chIRF7 polyclonal antibodies were obtained by using the recombinant proteins to produce mouse polyclonal mouse antiserum by immunizing mice; anti-TBK1 (ABGENT, Shanghai, China); anti-phospho-TBK1 (Cell Signaling, MA, USA); anti-HA and anti-Flag (Sigma, MO, USA); anti-HA and anti-Flag beads (Biotool, Houston, TX); and anti-β-tubulin (TransGen, Beijing, China). The blotted membranes were washed three times in TBST buffer and then incubated with 1:5,000 dilutions of horseradish peroxidase-conjugated secondary antibodies in TBST buffer for 1 h. The proteins were visualized using bioluminescence reagents (Pierce, Rockford, IL) in accordance to the manufacturer-specified protocol. The images were collected with a Tanon 5200 imaging system (Tanon, Shanghai, China).
9
Western Blot Analysis of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The si-NC and si-CHD5 siRNA were transfected into U87 cells with Lipofectamine 2000 reagent. After the cells were transfected for 48 h, Western blot analysis was performed as previously described [53 (link)]. Briefly, the cells were lysed with 1% SDS lysing buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Apexbio, Houston, TX, USA). The protein concentration was determined using the BCA protein assay reagent kit (Thermo Scientific, Waltham, MA, USA). All the blots were incubated with the respective primary antibodies, namely, anti-β-Tubulin (1:5000, TRANSGEN, Beijing, China), anti-CDK4, anti-CDK6, anti-CDK9 (1:800, Cell Signaling Technology, Boston, MA, USA), anti-E-cadherin, anti-N-cadherin, and anti-Twist1 (1:1000, Bioworld, Nanjing, China) antibodies. The protein bands were visualized with ECL Reagents (Smart-Lifesciences, Nanjing, China).
10
Autophagy and Cell Cycle Regulation Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The autophagy antibody sampler kit, the cell cycle regulation antibody sampler kit II, the AMPK and ACC antibody sampler kit, the antibody for 4E-BP1(#9644), phospho-4E-BP1(T37/46)(#2855), SQSTM1(#5114), S6K (#2708), phospho-S6K(#9234), phospho-pras40(T246)(#2997), the HRP-linked anti-rabbit and anti-mouse IgG antibodies were all from Cell signaling technology. The anti-Cyclin B1 (GNS1, sc-245) antibody was acquired from Santa Cruz and anti-β-Tubulin, anti-GAPDH and anti-β-Actin antibodies from Beijing TransGen Biotech (Beijing, China). The GlutaMAX supplement and puromycin dihydrochloride were from Gibco. The secondary fluorescently conjugated antibodies, anti-fade prolong Gold with DAPI were from Molecular Probes. Pre-stained Protein Ladder (26616) and M-PER buffer were from Thermo Pierce. Bafilomycin A1 was from Cayman. Chloroquine, NH4Cl, RO-3306 and Thymidine were from Sigma. Nocodazole was from Selleckchem. Protease inhibitor and Phosphatase inhibitor cocktails were from Roche and the PVDF membrane from Millipore.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!