The cells were treated with either PMGS (2 mg/mL) or PTX (50 nM) alone or in combination for 48 h, and then the proteins were extracted using a cell lysis buffer (P0013, Beyotime) and subsequently subjected to a Western blot analysis as described previously [30 (link)]. Briefly, equal amounts of proteins, which were quantified using a BCA Protein Assay Kit (P0010, Beyotime), were electrophoresed by SDS–PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (IPVH00010, Merck Millipore, Billerica, MA, USA). The membranes were blocked utilizing Protein Free Rapid Blocking Buffer (PS108P, Epizyme, Shanghai, China), incubated with appropriate primary antibodies at 4 °C overnight, and then exposed to HRP-conjugated secondary antibodies (Proteintech, Wuhan, Hubei, China) for 1 h at room temperature. The signals were acquired on a ChemiDoc XRS+ imaging system (Universal Hood Ⅱ, Bio-Rad, Hercules, CA, USA) with Image Lab software. GAPDH was used as the internal control. The antibodies used in this study are listed in Table S1 .
Protein free rapid blocking buffer
Manufactured by Ipsen
Sourced in China
Protein Free Rapid Blocking Buffer is a specialized buffer solution designed for use in various laboratory techniques that require efficient blocking of non-specific binding sites. The buffer formulation is free from proteins, making it suitable for applications where the presence of proteins could interfere with the desired outcome. The buffer is formulated to rapidly and effectively block non-specific binding sites, enabling more accurate and reliable results in downstream assays or experiments.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (8 mentions)
Ripa buffer, by Beyotime (4 mentions)
Bca protein assay kit, by Ipsen (4 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (3 mentions)
0.45 m nitrocellulose membrane, by Merck Group (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Omni ecl femto light chemiluminescence kit, by Ipsen (2 mentions)
Sds page, by Ipsen (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
P0013, by Beyotime (1 mentions)
Hrp conjugated secondary antibody, by Proteintech (1 mentions)
Chemi capture, by Clinx (1 mentions)
Chemiluminescent hrp substrate, by Ipsen (1 mentions)
Ripa buffer, by Ipsen (1 mentions)
Ecl kit, by GLPBIO (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
35 protocols using protein free rapid blocking buffer
1
Protein Extraction and Western Blot Analysis
2
Protein Expression Analysis in HEI-OC1 Cells and Basilar Membrane
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First, total protein was extracted from HEI-OC1 cells and basilar membrane by using RIPA buffer (Beyotime, Shanghai, China). Almost 10 μg of crude protein was denatured and electrophoresed on 12.5% SDS-PAGE gels. Proteins were transferred onto PVDF membranes by electro-blotting after electrophoretic separation, followed by blocking for 15 min at room temperature in Protein Free Rapid Blocking Buffer (Epizyme, Shanghai, China). The blots were incubated with SGK1, HIF-1α, P62, Bcl-2, Bax, Cleaved-Caspase3, and LC3B (information in Table 2 ) primary antibodies at 4°C overnight. After washing with PBS-T, membranes were hybridized with an appropriate secondary antibody (Abmart, Shanghai, China) at room temperature for 1 h. Lastly, images of the Western blot bands were performed with chemi Capture (Clinx, Shanghai, China) and the intensity in each group was measured with ImageJ. GAPDH was used as an internal control.
3
Western Blot Analysis of Protein Targets
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Western blot was carried out as described previously by us [3 (link), 26 (link)]. Briefly, the total proteins were collected from RIPA cell lysis by centrifugation. The proteins were denatured and separated by SDS-PAGE and subsequently transmitted into 0.22μm PVDF membrane. The membrane was blocked with Protein Free Rapid Blocking Buffer (Epizyme, Shanghai, China) for 20 minutes and subjected for antibody incubation overnight at 4° C. The follow antibodies were applied, including incubated with Rabbit anti-Flotillin-2(C42A3) (#3436, dilution 1:1000, CST, MA, USA), rabbit anti-BCAT1 antibody (D121976, dilution 1:200, BBI, Shanghai, China), rabbit anti-c-Myc (A1309, dilution 1:500, Abclonal, Wuhan, China), rabbit anti-p-AKT (Ser473)(D9E) (#4060, dilution 1:1000, CST, MA, USA), rabbit anti-AKT (#9272, dilution 1:1000, CST, MA, USA), rabbit anti-p-NF-κB p65 (Ser536)(93H1) (#3033, dilution 1:1000, CST, MA, USA), rabbit anti-NF-κB p65(D14E12) (#8242, dilution 1:1000, CST, MA, USA), and rabbit anti-GAPDH (AB-P-R001, dilution 1:1000, Goodhere, Hanzhou, China). Next day, after incubated with anti-rabbit or anti-mouse IgG HRP-conjugated secondary antibodies (D110058 or D110098, dilution 1:3000, BBI, Shanghai, China), the protein levels were visualized by chemiluminescent HRP substrate (EpiZyme, Shanghai, China).
4
Western Blot Protein Quantification Protocol
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In RAW264.7 cells, A549 cells, and lung tissues, proteins were exposed using RIPA buffer (EpiZyme, Shanghai, China). The protein concentration was detected with a BCA protein detection kit (Beyotime, Shanghai, China). Proteins were isolated from samples using SDS–PAGE. Protein-free rapid blocking buffer (EpiZyme, Shanghai, China) was used to block the PVDF membrane for 15 min upon transfer of the isolated protein into the PVDF membrane (Bio-Rad, CA, USA). The diluted primary antibody was reacted with PVDF membranes overnight at 4 °C. Next, the PVDF membrane was incubated at room temperature for 1 h with the corresponding secondary antibody (anti-rabbit or anti-mouse). Specific experimental conditions are shown in Supplementary Table 1 . Finally, proteins on PVDF membranes were imaged using an ultrasensitive ECL kit (GlpBio, USA) and protein expression was quantified using ImageJ.
5
Western Blot Analysis of Protein Markers
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Each group of cells was lysed and denatured using Cell Lysis Buffer for Western and IP (Beyotime Institute of Biotechnology), and protein quantification was performed by the BCA method. A total of 25 µg of protein sample per lane was denatured and electrophoresed using 10% SDS-PAGE, followed by semi-dry transfer of protein onto a PVDF membrane (Immobilion-P; EMD Millipore) and protein blocking with Protein Free Rapid Blocking Buffer (Epizyme) at room temperature for 30 min. The membrane was then incubated with the following primary antibodies at 4°C for 15 h: GAPDH (1:2,000 dilution; cat. no. AF1186; Beyotime Institute of Biotechnology), β-actin (1:2,000 dilution; cat. no. AF1186; Beyotime Institute of Biotechnology), RRM2 (1:1,000 dilution; cat. no. 11661-1-AP; Proteintech), β-catenin (1:1,000 dilution; cat. no. AC106; Beyotime Institute of Biotechnology), cyclin D1 (1:2,000 dilution; cat. no. AF0126; Beyotime Institute of Biotechnology) and c-MYC (1:2,000 dilution; cat. no. 10828-1-AP; Proteintech). Subsequently, it was incubated with HRP-labeled Goat Anti-Rabbit IgG (1:1,000 dilution; cat. no. A0208; Beyotime Institute of Biotechnology) for 1 h at room temperature and final visualization was performed using a GelDoc XR System (Bio-Rad Laboratories, Inc.). The experimental results were analyzed in grayscale using Image J (v1.8.0; National Institutes of Health).
6
Western Blot Analysis of CXCL12 and CXCR4
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Cells were washed twice with ice PBS, and total cell lysate samples were extracted and subjected to SDS-PAGE gel, then, transferred to a nitrocellulose membrane (Amersham, USA), and blocked with Protein-Free Rapid Blocking Buffer (Epizyme, China). The membranes were then reacted with primary antibodies including CXCL12 (Abcam9797, 1 : 1000), CXCR4 (SC53534, 1 : 1000), and β-actin (CST13E5, 1 : 1000) overnight at 4°C. After being washed with 1 × TBST buffer for three times, the membranes were incubated with secondary antibody (BIO-RAD, 1 : 3000, USA) for 1 hour at room temperature. The special bands from the conjugation reaction of protein antigen and antibodies were visualized by using Luminol from the enhanced chemiluminescence (ECL) kit (Santa Cruz, USA). Specific protein bands were measured with the densitometric intensity (SHST, China). The experiment above was repeated 3 times.
7
Protein Extraction and Western Blot Analysis
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Total protein was extracted and quantified using a total protein extraction kit (KeyGen Biotech, Nanjing, China) and BCA protein assay kit (EpiZyme, Shanghai, China), respectively, according to the manufacturer’s instructions. In total, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The PVDF membranes were blocked at room temperature for 30 min using protein-free rapid blocking buffer (EpiZyme), followed by the addition of primary antibody and incubation overnight at 4°C with gentle shaking. The primary antibodies included anti-actin beta (ACTB, 1 : 3000; 205 361-AP, Proteintech), anti-microtubule associated protein 1 light chain 3 beta (MAP1LC3B/LC3B, 1 : 1000; ab48394, Abcam, Cambridge, UK) and anti-p62/sequestosome 1 (SQSTM1, 1 : 10,000; ab56416, Abcam). The membranes were washed three times with TBST for 5 min and incubated with horseradish peroxidase-conjugated secondary antibodies (1 : 10,000; SA00001–2, Proteintech) for 2 h at 25°C. Finally, color development and quantification were performed.
8
Western Blot Analysis of Lung Proteins
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As reported previously [55 (link)], proteins in the lung or cells were extracted by RIPA lysis buffer containing protease and phosphatase inhibitors (Servicebio, China), and the protein concentration was determined using the BCA protein kit (Thermo, United States). After denaturation, proteins were separated by SDS-PAGE (Epizyme, China) and blotted onto activated PVDF membranes (Millipore, United States). After blocking with protein free rapid blocking buffer (Epizyme, China) for 10 min, the membranes were incubated with primary antibodies against GAPDH (1:50000, Proteintech), β-actin (1:50000, Proteintech), CDA1 (1:2000, Proteintech), α-SMA (1:1000, Abcam), collagen I (1:1000, Abcam), fibronectin (1:1000, Proteintech), TGF-β1 (1:1000, Proteintech), Smad3 (1:2000, Abcam), phosphorylated Smad3 (P-Smad3) (1:2000, Abcam), GFP (1:2000, Proteintech) at 4 °C overnight. After washing, the membranes were incubated with the appropriate secondary antibodies and visualized using an ECL Assay Kit (Epizyme, China). GAPDH/β-actin served as an internal control. The intensities of the target bands were quantified using ImageJ software.
9
Placental Protein Expression Analysis
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The protein levels of ALKBH5, MMP-2, MMP-9, and PLAC8 in placental tissue and trophoblast cells were measured by western blot. RIPA Lysis Buffer (Beyotime, CHN) containing protease inhibitors was used to cleave the placental tissue and trophoblast cells. The concentration of extracted protein samples were measured using a BCA kit (TheroFisher, USA). The samples were then transferred to a PVDF membrane after going through SDS-PAGE. Soon afterward, the membrane was blocked for 10 min with Protein Free Rapid Blocking Buffer (Epizyme, Shanghai, CHN). Subsequently, the membrane was incubated overnight at 4 °C with Anti-ALKBH5 antibody (ab195377; 1/1000), Plac8 (E1J2Z) Rabbit mAb (#13885; Cell Signaling), Anti-MMP2 antibody (ab92536; 1/2000), Anti-MMP9 antibody (ab76003; 1/2000), and Anti-Tubulin antibody (ab6160; 1/5000). Then, the membrane was incubated at room temperature with the secondary antibody before being immersed in enhanced chemiluminescence and exposed in the dark. The bands were observed with a chemiluminescence imager (TheroFisher, USA) [16 (link)].
10
Western Blot Analysis of Protein Targets
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Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer containing 1% phenylmethylsulfonyl fluoride (Beyotime, Shanghai, China) on ice for 30–60 min and centrifuged at 12,000 rpm for 5 min, and the pellet was discarded. The protein samples were boiled in sodium dodecyl sulfate (SDS)-loading dye for 15 min. The proteins were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto a 0.45-µm nitrocellulose membrane (Millipore, Bedford, MA, USA). The membranes were blocked with Protein Free Rapid Blocking Buffer (EpiZyme, Jiangsu, China) and subsequently probed with primary antibodies. The primary antibodies used were as follows: rabbit anti-human SET8 (#2996), anti-PIK3CB (#3011), anti-FOXO1 (#2880), anti-AKT (#4685), anti-p-AKT (#4060), and anti-BAK (#12105), anti-histone H4 (#13919), anti-BCL2 (#15071), and anti-GAPDH (#51332) purchased from Cell Signaling Technology (Beverly, MA, USA) and anti-H4K20me1 (Abcam; #ab177188; Cambridge, UK). Thereafter, the membranes were washed and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1.5 h at room temperature. Immunoreactive bands were visualized using an enhanced chemiluminescence detection system.
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