Washing of the treated cells was carried out in phosphate buffered saline (PBS) and lysed in RIPA buffer (high) (Solarbio, Beijing, China), which contained 1 mM phenylmethylsulfonyl fluoride, Solarbio. Calculation regarding protein concentration was done utilizing the bicinchoninic acid protein assay kit (Sangon Biotech). Equivalent amounts of whole protein extract (30 µg) were electrophoresed on SDS‐polyacrylamide gels and then transferred to polyvinylidene difluoride membranes (pore size: 0.22 µm) using the Yeasen Blot‐transfer unit. After blocking with 5% milk, incubated the membranes overnight with specific primary antibodies against Survivin (Mouse/IgG1; 1:3,000; Proteintech), glyceraldehyde‐3‐phosphate dehydrogenase (Mouse/IgG2b; 1:50,000; Proteintech), His (Mouse/IgG1; 1:10,000; Proteintech), Flag (Rabbit/IgG; 1:10,000; Bioworld), HA (Human influenza hemagglutinin) (Rabbit/IgG; 1:3,000; Bioworld), EGFP (Mouse/IgG1; 1:2000; Leading Biology) at 4°C. The membranes were later incubated with horseradish peroxidase‐conjugated secondary antibody (Goat Anti‐Rabbit IgG (H+L) or Goat Anti‐Mouse IgG (H+L); 1:6000; Proteintech) for 2 h on a horizontal oscillator at room temperature. Products were generated on film by the chemiluminescence kit purchased from Sangon Biotech and quantitated by ImageJ software.32 , 33 , 39
Ripa buffer high
Manufactured by Solarbio
Sourced in China
RIPA buffer (high) is a highly concentrated protein extraction and sample preparation solution. It is designed to efficiently lyse cells and solubilize proteins for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Solarbio (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bicinchoninic acid protein assay kit, by Sangon (1 mentions)
Chemiluminescence kit, by Sangon (1 mentions)
Goat anti mouse igg h l, by Proteintech (1 mentions)
Phenylmethylsulfonyl fluoride, by Solarbio (1 mentions)
Goat anti rabbit igg h l, by Proteintech (1 mentions)
Wblur0500, by Merck Group (1 mentions)
Anti ki67, by Abcam (1 mentions)
Anti β actin, by Abcam (1 mentions)
Anti tsg101, by Abcam (1 mentions)
Anti cd81, by Abcam (1 mentions)
Anti pcna, by Abcam (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Zhongshan Golden Bridge Biotechnology (1 mentions)
P8340, by Merck Group (1 mentions)
Hy k0023, by MedChemExpress (1 mentions)
Hy k0022, by MedChemExpress (1 mentions)
P pi3k, by Cell Signaling Technology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
13 protocols using ripa buffer high
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Protein Expression
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Cells and lung tissue lysates were extracted with RIPA buffer (high) (Solarbio, R0010, China) containing 1% protease inhibitors (MCE, USA) and a phosphatase inhibitor cocktail. The samples were mixed with SDS lysis buffer (10% SDS, 0.25 M Tris-HCl at pH 6.8, 50% glycerol, 1% β-mercaptoethanol, and bromophenol blue) and separated by SDS‒PAGE. For nonreducing gel analysis, the cells were lysed in 20 mM Tris (pH 7.0) containing 0.5% NP40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, 20 mM glycerol phosphate, 1 mM sodium vanadate, and 1 μg/mL leupeptin and separated by native PAGE. Then, the proteins were transferred to PVDF membranes (Millipore, USA). After being blocked with 5% skim milk or BSA for 1 h, the membranes were incubated with primary antibodies at 4 °C overnight and then with the HRP-labeled secondary antibodies for 1 h at room temperature. The antibodies used for protein expression analysis are listed in Supplementary Table 2 . Bands were visualized with an enhanced chemiluminescence kit (WBLUR0500, Millipore, USA).
3
Exosomal Protein Extraction and Analysis
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As previous study [16 (link)], the proteins were extracted from NHLF and the exosomes from A549 cells using RIPA buffer (high) (R0010, Solarbio, Shanghai, China) on ice for 20 min and quantified by BCA Kit (PC0020; Shanghai Acmec Biochemical Co., Ltd., Shanghai, China). All antibodies were purchased from Finetest Biotech Ltd. (Wuhan, China) or Abcam (Shanghai, China): anti-CD81 (1:500; FNab10448), anti-TSG101 (1:1000; FNab10812), anti-Alix (1:1000; ab275377), anti-tubulin (1:5000; ab6160), anti-Ki67 (1:200; FNab09788), anti-PCNA (1:5000; FNab06217), anti-Bcl-2 (1:1500, ab194583), anti-Bax (1:1500, ab32503), anti-Caspase-3 (1:2000, ab2302), anti-SPRY1 (1:1000; ab111523), and anti-β-actin (1:5000; ab8226). β-actin was used to normalize protein expression levels. Afterward, the horseradish peroxidase-labeled goat anti-rabbit HRP antibody (1:5000, ab97051) and goat anti-mouse HRP antibody (1:5000, ab205719) were purchased as the secondary antibody. Finally, protein bands were developed using ECL Plus Ultra-Sensitive Kit (PH0353, Phygene Life Sciences Company, Fuzhou, China).
4
Western Blotting of Cell Signaling Proteins
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Western blotting was performed as described by Yang et al. (Yang et al., 2012 (link)). Briefly, cells were washed with cold PBS and lysed in radioimmunoprecipitation assay (RIPA) buffer (high) (R0010, Solarbio) containing a protease inhibitor cocktail (P8340, Merck Millipore) and phosphatase inhibitor cocktails II and III (HY-K0022, HY-K0023, MedChem Express). Thirty micrograms of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) followed by electrotransfer onto nitrocellulose membranes. After blocking with 5% skim milk for 1 h, membranes were incubated with primary antibodies against proliferating cell nuclear antigen G-CSF (1:200; #bs-1023R; Bioss), G-CSFR (1:300; #bs-2574R; Bioss), p-PI3K (1:300; #4228; Cell Signaling Technology), PI3K (1:300; #4249; Cell Signaling Technology), p-Akt (1:300; #4060; Cell Signaling Technology), Akt (1:500; #4691; Cell Signaling Technology), or GAPDH (1:2000; #ab22556; Abcam) overnight at 4°C. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (Zhongshan Golden Bridge Biotechnology Co., Ltd) for 1 h at room temperature, and signals were developed using an enhanced chemiluminescence system (Piece). Densitometric quantification was performed using Scion Image software (Scion Corp.).
5
Probiotic and β-Glucan Modulate Colitis
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Fubon feed additive and S. boulardii β-glucan (purity 80%, molecular mass 800–1000 kDa) were purchased from Angel Yeast Co., Ltd. (Yichang, China). Yeast Extract Peptone Dextrose Medium (YPD) was purchased from Qingdao Hi-Tech Park Haibo Biotechnology. Dextran sulfate sodium (DSS, w/v. molecular mass 36–50 kDa) was purchased from Yisheng Biotechnology Co., Ltd. (Shanghai, China). RIPA buffer (high) and PMSF were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Protease inhibitor, ZO-1 Rabbit Polyclonal Antibody, Occludin Rabbit Polyclonal Antibody, GAPDH Rabbit Polyclonal Antibody, Horseradish peroxidase-conjugated goat anti-rabbit IgG(H + L) secondary antibody, and an ELISA Kit (Mouse TNF-α, IL-1β, and IL-6) were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). TransZolTM Up Plus RNA Kit and Reverse transcriptase reagent kit were purchased from Beijing TransGen Biotechnology Co., Ltd. (Beijing, China). SuperReal PreMix Plus (SYBR Green) was purchased from Tiangen Biochemical Technology Co., Ltd. (Beijing, China).
6
Exosome Protein Extraction and Quantification
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The protein samples of the cells and exosomes were collected, lysed in RIPA buffer (high) (R0010, Solarbio, Shanghai, China) on ice for 20 min, and quantified by BCA protein assay kit (PC0020, Solarbio, Shanghai, China). In our work, all antibodies were purchased from Abcam (Shanghai, China) and Proteintech (Wuhan, China). TSG101 (ab125011), CD63 (ab134045), Grp94 (ab238126), MMP‐2 (ab181286), MMP‐9 (ab137867), N‐cadherin (22018‐1‐AP), and vimentin (10366‐1‐AP) antibodies were in 1:2000 dilutions. Cytochrome C (ab133504), E‐cadherin (20874‐1‐AP) (ab2324), and β‐actin (66009‐1‐Ig) antibodies were in 1:4000 dilutions. The horseradish‐peroxidase labeled goat anti‐rabbit (SA00001‐2) or goat anti‐mouse (SA00001‐1) antibodies were 1:5000 dilution as the secondary antibody. The visualization of protein bands was performed by ECL western blotting substrate (PE0010, Solarbio, Shanghai, China), and the intensities of protein bands were quantified by Image J software (ImageJ 1.51j8, National Institutes of Health, USA).
7
Hippocampal RNA and Protein Isolation
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Cell pellets were taken at 24 hours after drug treatment; the rats were killed at 26 days old and the hippocampus were dissected on ice, snap-frozen in liquid nitrogen immediately. RNA and miRNA were isolated from the same cell pellet and hippocampus tissue using miRcute-miRNA Extraction and Isolation kit (TIANGEN, china) following the manufacturer’s protocol. Proteins were isolated from another cell pellet and hippocampus tissue by using RIPA buffer (high) (Solarbio). RNA quality was checked on a denaturing RNA Ethidium bromide (EB) gel (1% agarose, 110 V, 30 min). After proteins were isolated already, the protein content was determined by using BCA Protein Assay Kit (Solarbio).
8
Corneal Protein Expression Analysis
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Corneal tissues of four eyes were collected and pooled as one sample, and three samples were used in each group. Tissue samples were lysed and the protein samples were extracted in RIPA buffer (high) (Solarbio) containing protease inhibitors. Sample protein concentrations were determined by a BCA Protein Assay Kit (Beyotime). Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane, which was incubated overnight with the appropriate primary antibody. Antibodies specific for MMP-2, MMP-9, SOD-1, and COX-2 were used: anti-MMP-2 (1:1000, Proteintech, 10373-2-AP), anti-MMP-9 (1:1000, Proteintech, 10375-2-AP), anti-SOD-1 (1:1000, Proteintech, 10269-1-AP), anti-COX-2 (1:1000, Proteintech, 12375-1-AP), and β-actin antibody (1: 10,000, Proteintech, 20536-1-AP). The secondary antibody used was HRP-conjugated goat anti-rabbit IgG (1:1000, Proteintech, SA00001-2). The bands were visualized by enzyme-linked chemiluminescence using the ECL kit (Vazyme), and the image intensity was calculated with ImageJ Software.
9
Western Blot Analysis of PCNA Protein
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The protein of PCNA in the normal and tumor tissues were analyzed by Western blot. Briefly, the tissues were treated with RIPA buffer (high) (Solarbio, Beijing, China) in ice for 10min and were lysed with pestle in ice for 5 min. Total lysates were centrifuged and the protein concentration of the supernatant was tested with a BCA protein assay kit (Beyondtime, Nantong, China). 20 μg of total proteins were separated by SDS-PAGE and electro-blotted onto a PVDF membrane (Merck KGaA). The membranes were blocked with 3% BSA for 1 h at room temperature, and incubated with different primary antibodies: anti-PCNA (#ab18197; Abcam, Cambridge, UK) and anti-GAPDH (#ab181602; Abcam). After incubated with the appropriate secondary antibodies for 1 h at room temperature, the bands were visualized using an ECL assay kit (Amersham Pharmacia Biosciences) and observed with an LAS3000® Luminescent image analyzer.
10
Quantification of Membrane Protein Expression
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The membrane proteins of each tissue were extracted with RIPA buffer (high) (Cat No. R0010, Solarbio, Beijing, China). The concentration of protein was measured with a Protein Quantitative Reagent Kit-BCA Method. Protein samples (extracted from mantle or gill at the same time point) were mixed depending on their concentration and then subjected to SDS-PAGE; the separated proteins were then electrophoretically transferred onto a PVDF membrane. In brief, the membrane was blocked with 5% skim milk in TBST at room temperature for 1 h. Then, the blots were incubated with primary antibodies against the NKA α subunit (1:500, ATP1A2 Rabbit Polyclonal, Cat. No. 16836-1-AP, Proteintech, Rosemont, IL, United States) overnight at 4°C, followed by incubation with appropriate peroxidase-conjugated secondary antibodies (1:3000, HRP Goat Anti-Rabbit IgG (H+L), Cat. No. AS014, ABclonal, Wuhan, China). Gray values were analyzed using ImageJ software. Actb served as an internal control (1:1000, ACTB Monoclonal Antibody, Cat. No. AC026, ABclonal, Wuhan, China), and the initial group (0 h) was used as the normalized group. The detail information and the specificity of primary antibody against HdNKA α and actb were shown in Supplementary Figure S1 .
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