Immunoblotting was performed as described previously [5 (link)]. Briefly, LV tissue was homogenized in ice-cold lysis buffer. Crude homogenates were centrifuged for 5 min at 10000 g, and protein concentration of the supernatants was determined by the BCA method (Pierce Chemical Co). Supernatants were added to loading buffer and heat denatured by boiling for 3 min. Protein (25 ug) was electrophoresed in separate lanes on 8% SDS-PAGE. After electrophoresis, proteins were transferred onto nitrocellulose membranes and blocked in a 5% nonfat dried milk solution at room temperature. The blot was incubated with antibodies against collagen I or collagen III (1 : 100 dilution; Beijing Biosynthesis Biotechnology Company, China). A peroxidase-conjugated avidin secondary antibody was applied for visualization (1 : 5000 dilution; Santa Cruz Biotechnology, USA). The blots were stripped and reprobed with a β-actin antibody (1 : 2000; Beijing Biosynthesis Biotechnology Company, China) to normalize for protein loading. The integrative grayscale pixel area-density was captured with a CCD camera and quantified using Quantity One software.
β actin antibody
Manufactured by Bioss Antibodies
Sourced in China, United States, United Kingdom
The β-actin antibody is a primary antibody that recognizes the β-actin protein, a ubiquitously expressed cytoskeletal protein. It can be used to detect and quantify the expression of β-actin in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
P akt, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Abcam (2 mentions)
Bca method, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Bioss Antibodies (1 mentions)
Las 4000, by Fujifilm (1 mentions)
Bradford protein assay kit, by Bio-Rad (1 mentions)
Affinipure goat anti rabbit igg, by ZSGB-BIO (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Parp asp214, by Cell Signaling Technology (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Tb green premix ex taq, by Takara Bio (1 mentions)
Vav3 antibody, by Abcam (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Proteintech (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Kn 62, by Bio-Techne (1 mentions)
Phospho pka c, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Pgc 1α, by Abcam (1 mentions)
22 protocols using β actin antibody
1
Immunoblotting for Collagen I and III
2
Protein Extraction and Western Blot
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Part (100 mg) of the collected tissues was cut into pieces and homogenized in cold lysis buffer containing protease inhibitor. Centrifugal separation was conducted, at 4°C, at 10,000 rpm for 5 minutes. The upper layer of the solution was tested for protein using the Bradford method.8 (link) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed. The primary antibody was added (rabbit antibody TNF-α [diluted 1:500]; rabbit antibody E-cadherin [diluted 1:500]; and β-actin antibody [diluted 1:1,000] [Beijing Biosynthesis Biotechnology Co., Ltd, Beijing, People’s Republic of China]), and the nitrocellulose filter membrane was incubated at 4°C throughout the night, then was washed prior to incubation with the secondary antibody (diluted at 1:1,000), and finally, marked by horseradish peroxidase at 37°C for 1 hour. Quantification was performed with Gel-Pro Analyzer software version 4 (Media Cybernetics, Rockville, MD, USA).
3
Antibody-based Characterization of MRSA
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LZD was obtained from Beijing Solarbio Science & Technology Co. Ltd (Beijing, People’s Republic of China). The CD63 and flotillin 1 antibodies were purchased from Abcam Biotechnology (Cambridge, MA, USA). The β-actin antibody and goat-anti-rabbit horseradish-peroxidase (HRP)-conjugated secondary antibody were purchased from Beijing Biosynthesis Biotechnology Co. Ltd (Beijing, People’s Republic of China). The MRSA strain WHO-2 (WHO-2) was kindly provided by Rongxin Qin (Army Medical University, Chongqing, People’s Republic of China).
4
Protein Extraction and Western Blot Analysis
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The cells were washed twice with phosphate-buffered saline and lysed directly in lysis buffer (1 mmol/l PMSF, 1 mmol/l EDTA, 40 mmol/l Tris-HCl, 100 mmol/l NaVO3, 150 mmol/l KCl and 1% Triton X-100) on ice for 15 min. Subsequent to centrifugation (12,000 × g for 20 min at 4°C), the Bradford protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to measure the protein concentrations of the lysates. Equal amounts of proteins were separated on 10% SDS-PAGE gels and then electrotransferred onto a polyvinylidene difluoride membrane. The membranes were blocked in 5% skimmed milk-Tris-buffered saline plus Tween-20 solution and incubated with rabbit MMP14 monoclonal antibody (1:1,000; Abcam, Cambridge, UK) or rabbit polyclonal β-actin antibody (1:1,000; Bioss, Beijing, China). Following incubation with peroxidase-conjugated AffiniPure goat anti-rabbit IgG (1:2,000; ZSGB-BIO, Beijing, China), protein bands were detected with Fujifilm Las-4000 (Fujifilm, Tokyo, Japan).
5
Investigating Drug-Resistant Ovarian Cancer
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The drug‐resistant ovarian cancer cell line, SKOV3/DDP, was bought from the Institute of Cancer Research, Chinese Academy of Medical Sciences (Beijing, China).19 , 20 Cell counting kit‐8 (CCK‐8) and Annexin V‐FITC were purchased from Jiamay Biotech (The catalog numbers are AP1008 and LHK601‐100, respectively; China). Primary antibodies against FOXO3a or PUMA were purchased from Abcam (The catalog numbers are ab12162 and ab9643, respectively; USA). β‐actin antibody was purchased from Bioss (Catalog number: bs‐0061R; China). Plasmids GFP‐Foxo3a and GFP‐vector were purchased from Gene (Shanghai, China). Lipofectamine 2000 was purchased from Invitrogen (Catalog number: 11668019; USA).
6
Western Blot Analysis of Protein Signaling
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SDS buffer (60 mM Tris-HCl with a pH value at 6.8, 10% glycerol, 2% SDS, with 5% 2-mercaptoethanol) was utilized to store the lysate of the cells and tissues. The 4–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels were used to separate the cell lysate. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Invitrogen), and the membranes were blocked with Tris-buffered saline plus 0.1% Tween-20 (TTBS) containing 5% skim milk for 2 h. The primary antibodies specific to phospho-ERK, total-ERK, phospho-4E-BP1, phospho-mTOR, total-mTOR, phospho-AMPK, total-AMPK, poly(ADP-ribose) polymerase (PARP) (Asp214) (Cell Signaling Technology, Beverly, MA, USA), Bcl-2 (Abgent, Inc., San Diego, CA, USA) were immunoblotted. All bands were washed in TBS with Tween-20 (TBST) and immunoblotted with peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies, respectively. The bands were detected using an enhanced chemiluminescence (ECL) Western Blotting kit and exposed to film. The β-actin antibody (BIOSS, Beijing, China) was used as an internal marker for control. All experiments were carried out thrice.
7
Mitochondrial Protein Expression Analysis
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Cytochrome c (Cyt C) antibody was purchased from Boster (Wuhan, China). TGF-β antibody, collagen I antibody, PPARγ antibody, ATP synthase β (ATP β) antibody, Cyt C oxidase subunit I (COX I) antibody, NDUFB8 antibody, optic atrophy 1 (Opa1) antibody, mitofusin (Mfn)2 antibody, dynamin-related protein 1 (Drp1) antibody, voltage-dependent anion-selective channel (VDAC) antibody and β-actin antibody were purchased from Bioss (Beijing, China). Fibronectin antibody was purchased from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China).
8
Characterizing Molecular Mechanisms in Cells
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JS-K was purchased from Santa Cruz Biotechnology (California, USA); Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (DMEM/F12) was obtained from Hyclone (Utah, USA); Fetal bovine serum (FBS) was purchased from Gibco (California, USA); USP13 and TAGLN antibodies were purchased from Santa Cruz Biotechnology (California, USA); β-actin antibody was purchased from BIOSS (Beijing, China); ECL reagents were purchased from Solarbi (Beijing, China); PrimeScript RT reagent Kit and TB Green Premix Ex Taq were purchased from Takara (Tokyo, Japan). CCK8 reagent was purchased from Living (Beijing, China).
9
Western Blot Analysis of VAV Proteins
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The following antibodies were used: VAV1 antibody (Abcam, Cambridge, UK), VAV2
antibody (Abcam, Cambridge, UK), VAV3 antibody (Abcam, Cambridge, UK), β-actin
antibody (Bioss, Massachusetts, USA), and horseradish peroxidase (HRP)-linked
anti-rabbit immunoglobulin G (IgG) (CST, Massachusetts, USA). The reagent was
RIPA Lysis Buffer (Biyuntian Biotechnology, Shanghai, China).
antibody (Abcam, Cambridge, UK), VAV3 antibody (Abcam, Cambridge, UK), β-actin
antibody (Bioss, Massachusetts, USA), and horseradish peroxidase (HRP)-linked
anti-rabbit immunoglobulin G (IgG) (CST, Massachusetts, USA). The reagent was
RIPA Lysis Buffer (Biyuntian Biotechnology, Shanghai, China).
10
Evaluating Protein Expression in A549 Cells
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Total protein was extracted from the A549 cells and quantified using the BCA protein assay kit (Solarbio, Beijing, China). A sample containing 20 μg of protein was separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (Solarbio), transferred to a polyvinylidene fluoride membrane, and incubated with rabbit anti-mouse N-cadherin, GRP78, and α-SMA primary antibodies (Proteintech, Wuhan, China), or β-actin antibody (Bioss, Beijing, China) as a loading control, at room temperature for 2 h. Subsequently, the membrane was washed, incubated with the horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Proteintech, Wuhan, China) for 1 h at room temperature, and exposed using the ECL kit. The experiment was repeated three times.
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