Total protein content was extracted from SH-SY5Y cells or EVs using RIPA kits (R0010, Solarbio, Beijing, China). Briefly, 40 µg of each sample was extracted, separated with 10% SDS-PAGE, and electrotransferred onto a PVDF membrane (Merck Millipore, Billerica, MA, USA). Subsequently, the membrane was blocked and incubated with the following primary antibodies: EGR1 (dilution ratio of 1:1000, #4154, CST, Beverly, MA, USA), NOX4 (dilution ratio of 1:2000, ab109225, Abcam), p–p38 (dilution ratio of 1:1000, #4511, CST), P38 (dilution ratio of 1:1000, #8690, CST), TH (dilution ratio of 1:5000, ab137869, Abcam), 4-HNE (dilution ratio of 1:1000, ab46545, Abcam), and GAPDH (dilution ratio of 1:10000, ab128915, Abcam). Afterward, the membrane was incubated with IgG (dilution ratio of 1:10000, ab97051, Abcam). Later, the membrane was developed using an electrogenerated ECL, and then the membrane was exposed to light using the Image Quant LAS 4000 C gel imager (GE Company, Schenectady, NY, USA) and analyzed with the ImageJ software (1.48 u, National Institutes of Health, Maryland).
Ripa kit
Manufactured by Solarbio
Sourced in China
The RIPA kit is a laboratory equipment used for protein extraction and preparation. It contains a buffer solution and reagents designed to lyse cells and solubilize proteins for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Ab128915, by Abcam (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Ab32503, by Abcam (2 mentions)
Ab205718, by Abcam (2 mentions)
Ecl808 25, by Biomiga (2 mentions)
Ab109225, by Abcam (1 mentions)
Ab137869, by Abcam (1 mentions)
Ab97051, by Abcam (1 mentions)
Ab46545, by Abcam (1 mentions)
Adcy1, by Abcam (1 mentions)
Ab97779, by Abcam (1 mentions)
Ab76003, by Abcam (1 mentions)
Ab205718, by Solarbio (1 mentions)
Ab92552, by Abcam (1 mentions)
Ab174314, by Abcam (1 mentions)
Ab92742, by Abcam (1 mentions)
Ab32042, by Abcam (1 mentions)
Ab59348, by Abcam (1 mentions)
Ecl reagent, by Solarbio (1 mentions)
14 protocols using ripa kit
1
Protein Quantification and Analysis in EV Samples
2
Quantification and Characterization of Stem Cell Markers
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Total protein content was obtained from the lysed cells using a radio-immunoprecipitation assay (RIPA) kits (R0010, Solarbio, Beijing, China) and then quantified using a bicinchoninic acid (BCA) quantitative kit (G3522-1, Guangzhou Jabes Biotechnology Co., Ltd., Guangzhou, China). Next, the protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. After being blocked 5% bovine serum albumin (BSA) at room temperature for 1 h, the membrane was incubated with diluted primary antibodies purchased from Abcam, Cambridge, MA, USA: ADCY1 (1 : 10000, ab203204), CD133 (1 : 1000, ab216323), CD44 (1 : 1000, ab157107), leucine-rich G protein-coupled receptor-5 (LGR5) (1 : 1000, ab75732), and aldehyde dehydrogenase isoform 1 (ALDH1) (1 : 500, ab129815) overnight at 4° C. Next, the membrane was incubated with the horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (1: 5000, Beijing Zhongshan Biotechnology Co. Ltd., Beijing, China). Finally, an enhanced chemiluminescence (ECL) reagent kit was employed to visualize the results after which the gray value was calculated, with GAPDH regarded as the internal control. The cellular experiment was repeated three times to obtain the mean value.
3
Quantitative Western Blot Analysis Protocol
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A radioimmunoprecipitation assay (RIPA) kit (Solarbio) was used to extract total proteins. Then, proteins were separated, electro-transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad) and blocked with skim milk. Subsequently, membranes were incubated with the corresponding primary antibodies (Abcam, Cambridge, MA, USA), including an anti-proliferating cell nuclear antigen (anti-PCNA; ab92552), anti-Ki-67 (ab92742), anti-B-cell lymphoma-2 (anti-Bcl-2; ab59348), anti-cleaved caspase 3 (anti-c-caspase 3; ab32042), anti-caspase 3 (ab13847), anti-metalloproteinase-2 (anti-MMP2; ab97779), anti-metalloproteinase-9 (MMP9; ab76003), anti-PAPPA (ab174314) and anti-GAPDH (ab9485), at 4°Covernight. Next, the secondary antibody (goat antirabbit; ab205718) was added for incubation at room temperature for 1.5 h. Finally, an enhanced chemiluminescence (ECL) reagent (Solarbio) was used to visualize the protein blots.
4
Protein Extraction and Western Blot Analysis
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Total protein was extracted from OS tissue using a RIPA kit (R0010, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The total protein concentration was estimated using a BCA protein assay kit (69-21875, Wuhan Mokesha Biotech Company, Hubei, China). Proteins were separated by 10% SDS-PAGE (20050227, Beyotime Biotechnology Co., Shanghai, China) and transferred onto membranes. The membranes were incubated overnight at 4°C with HOXA9 (1: 2000, ab140631), Wnt (1: 3000, ab28472), β-catenin antibody (1: 5000, ab32572), WIF-1 (1: 2000, ab171766), Survivin (1: 10000, ab76424), Cyclin D1 (1: 10000, ab134175), c-Myc (1: 10000, ab166837), BIM (1:2000, ab32158), Bax (1: 2000, ab32503), Mcl-1 (1: 1000, ab32087), Bcl-xL (1: 2000, ab32503), or Snail (1: 500, ab53519) rabbit anti-human antibodies, which were purchased from Abcam (Cambridge, MA, USA). Membranes were then incubated with an HRP-conjugated goat anti-rabbit IgG secondary antibody (1: 1000, BA1056, Wuhan Boster Biological Technology Co., LTD., Hubei, China) or an HRP-conjugated goat anti-mouse IgG antibody (1: 5000; Zhongshan Biotech Ltd., Beijing, China). Blots were developed in the dark using the ECL reagent (WBKLS0500, Pierce, Rockford, IL, USA) and imaged.
5
Protein Expression Analysis from Cells
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Total protein from tissues and cells was extracted using radio-immunoprecipitation assay (RIPA) kit (Beijing Solarbio Science and Technology Co., Ltd, Beijing, China). Protein concentration was determined using a BCA protein assay kit (GBCBIO Technologies Inc., Guangzhou, China). The proteins were subsequently separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Following separation, the proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Billerica, MA, USA), and subsequently blocked for 2 h with Tris Buffered Saline with Tween (TBS/T) containing 5% skimmed milk. The PVDF membranes were incubated with primary antibody β-Tubulin (ab210797, 1:1000, Rabbit), TPM1 (ab38898, 1: 1000, Rabbit), CD63 (ab216130, 1:1000, Rabbit), HSP70 (ab181606, 1:1000, Rabbit), and Calnexin (ab22595, 1:1000, Rabbit) overnight at 4 °C. The membrane was then incubated with the secondary antibody goat anti-rabbit immunoglobulin G (IgG) (ab150077, 1:1000, Abcam, UK) at room temperature. Enhanced chemiluminescence (ECL) was applied for visualization. The image was subjected to gray scale analysis using Image J software.
6
Protein Expression Analysis Protocol
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Total protein was extracted from the EC cell lines using a radioimmunoprecipitation assay (RIPA) kit (Solarbio). The protein samples were separated by 10% SDS–PAGE (Sangon Biotech, Shanghai, China) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Boston, USA) according to a standard process. After blocking in 5% skim milk (BD Difco, Maryland, USA) or 5% bovine serum albumin(BSA, Biofroxx, Einhausen, Germany) for 2 h, the membranes were incubated overnight at 4 oC with primary antibodies against DCLK1 (JA11-03, HUABIO, Hangzhou, China), E-cadherin (ab40772, Abcam, Cambridge, UK), N-cadherin (ab76011, Abcam), Cyclin D1 (26,939-1-AP, Proteintech, Wuhan, China), CDK4 (#12,790, Cell Signaling Technology, Massachusetts, USA), PI3K p85 (ab191606, Abcam), p-PI3K p85 (phospho Y607) (ab182651, Abcam), Akt (60,203-2-lg, Proteintech), p-Akt (phospho Ser 473) (66,444-1-lg, Proteintech), NF-κB p65 (ab76311, Abcam), or NF-κB p-p65 (phospho S536) (ab76302, Abcam), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibodies(LF101 or LF102, Epizyme, Shanghai, China) for 2 h (1:5000). β-actin (AP0060, Bioworld, Nanjing, China) or GAPDH (AP0063, Bioworld) antibodies were used as the internal controls at a dilution of 1:5000. The protein signals were analyzed using the enhanced chemiluminescence method (Millipore).
7
Western Blot Protein Analysis Protocol
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Total protein was extracted from cells using a radioimmunoprecipitation assay (RIPA) kit (R0010, Beijing Solarbio Science & Technology Co. Ltd., Beijing, China). After quantifying total protein concentration by a bicinchoninic acid (BCA) method, proteins were separated by 12% SDS-PAGE and then transferred onto nitrocellulose membranes. Subsequently, the membranes were sealed by 5% nonfat milk for 1 h and then incubated overnight at 4°C by primary antibodies. On the next day, the membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibody (Santa Cruz, CA, USA) after being washed by PBS for three times. Finally, the protein bands were then measured with an enhanced chemiluminescence detection kit (KeyGen, Nanjing, China).
8
Protein Expression Analysis Protocol
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Total cellular protein was extracted using the total protein extraction solution RIPA kit (R0010; Solarbio, China). The protein concentration was determined according to the instructions of BCA protein quantitative kit. The protein concentration was calculated according to the standard curve. The protein was separated by 10% SDS-PAGE electrophoresis. Proteins are electrostatically transferred to the NC membrane. Substrate chromogenesis or radioautography was used to detect the protein component expressed by a specific target gene, separated by electrophoresis. The primary antibodies that were used were rabbit anti-N-cadherin (1: 5000, 22018-1-AP; Proteintech), rabbit anti-E-cadherin (1: 50000, 20874-1-AP; Proteintech), rabbit antivimentin (1: 5000, 10366-1-AP; Proteintech), rabbit anti-SOX2 (1: 600, 11064-1-AP; Proteintech), rabbit anti-CD133 (1: 800, 18495-1-AP; Proteintech), rabbit anti-PPAT (1: 1000, 15401-1-AP; Proteintech), and rabbit anti-IMPDH1 (1: 5000, 22092-1-AP; Proteintech). This was followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (1: 5000, SA00001-2; Proteintech). The membrane was immersed in SuperECL Plus (K-12045-D50, Advansta, USA) for luminescence visualization using β-actin as an internal reference.
9
Protein Extraction and Quantification in HCC Cells
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Total protein in HCC cells was extracted by the RIPA kit (R0010, Beijing Solarbio Life Sciences Co., Ltd., Beijing, China). Total protein was separated by 10% sodium dodecyl sulphatepolyacrylamide gels electrophoresis (SDS-PAGE), which then was transferred onto the polyvinylidene difluoride (PVDF) membranes (Millipore Corp., USA). The membranes loaded with protein shaken for blockage 2h with 5% skim milk at room temperature. Subsequently, the PVDF membranes were incubated with anti-SSRP1 polyclonal antibody (ab26212, 1:1500 dilution, Abcam, Cambridge, UK, USA) and mouse against GAPDH antibody (ab128915; 1:1500 dilution; Abcam, Cambridge, UK, USA) overnight at 4°C, respectively. After that, we continued to incubate the PVDF membranes with horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibodies (ab205718; 1:5000 dilution; Abcam, Cambridge, UK, USA) at room temperature for 1h. The Enhanced Chemiluminescence Reagent (Bio-Rad Labora-tories, Hercules, CA, USA) were used to visualize the immunoreactive bands.
10
Western Blot Analysis of Apoptosis Markers
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Total protein was extracted using RIPA kit (R0010, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Next, protein was separated using 10% sulfate polyacrylamide gel electrophoresis gel (SDS/PAGE), and transferred on to a polyvinylidene fluoride (PVDF) membrane which was then blocked with Tris-buffered saline with Tween 20 (TBST) solution containing 5% bovine serum albumin (BSA). After that, the membrane was incubated with the following primary rabbit polyclonal antibodies to BCL2-associated X (Bax; 1:1000, ab32503), B-cell lymphoma-2 (Bcl-2; 1:1000, ab32124), Caspase 3 (1:500, ab4051), Cleaved Caspase3 (1:500, ab2302), CyclinD1 (1:1000, ab134175) and GAPDH (1:100, ab37168) overnight at 4°C. The antibodies were all from Abcam Inc. (Cambridge, U.K.). Then, the membrane was incubated with the secondary goat anti-rabbit antibody to IgG (1:5000, Beijing Zhongshan Biotechnology Co., Ltd., Beijing, China). Blots were visualized using electrochemiluminescence (ECL) chromogenic substrate.
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