The L3–L5 DRG tissue was separated and homogenized in RIPA cleavage buffer (P0013, Beyotime, Shanghai, China) containing protease and phosphatase inhibitors (4693132001, Roche, Germany). The protein concentration was determined using a BCA analysis kit (23225, Thermo Fisher Scientific, Waltham, MA, USA). The 30μg protein samples were loaded into the SDS–PAGE gel sample pore and then transferred to the 0.22 μm PVDF membrane. Then, the membrane was blocked with 5% no-fat milk and incubated overnight at 4 °C with antibodies against γH2AX (1:500), H2AX (1:500), H3 (rabbit, 1:200, Cell Signaling Technology, Boston, MA, USA, 7631), ERK (rabbit, 1:1000, Cell Signaling Technology, Boston, MA, USA, 9102S), pERK (rabbit, 1:1000, Cell Signaling Technology, Boston, MA, USA, 9101S), AKT (rabbit, 1:1000, Cell Signaling Technology, Boston, MA, USA, 4691), and pAKT (rabbit, 1:1000, Cell Signaling Technology, Boston, MA, USA, 4060). The images were captured using an Odyssey Imaging System (LI-COR Bioscience, Lincoln, Northeast, Dearborn, MI, USA). ImageJ software was used to analyze the intensity of the selected strip.
Ripa cleavage buffer
Manufactured by Beyotime
Sourced in China
RIPA cleavage buffer is a protein extraction buffer used to lyse cells and extract total cellular proteins. It contains a combination of ionic and non-ionic detergents that solubilize cellular membranes and denature proteins, allowing the extraction of a wide range of proteins from various cell types and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Beyotime (2 mentions)
Tbst solution, by Solarbio (2 mentions)
Ecl kit, by Solarbio (2 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Bca analysis kit, by Thermo Fisher Scientific (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Phenylmethanesulfonylfluoride fluoride, by Beyotime (1 mentions)
0.45 μm polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ecl kit, by Beyotime (1 mentions)
Enhanced chemi luminescence (ecl), by Beyotime (1 mentions)
Sds page gel, by Beyotime (1 mentions)
Gapdh, by Proteintech (1 mentions)
Ab38449, by Abcam (1 mentions)
Ab134903, by Abcam (1 mentions)
Ab207612, by Abcam (1 mentions)
Ab278545, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Ab192890, by Abcam (1 mentions)
7 protocols using ripa cleavage buffer
1
Analyzing DRG Protein Expression
2
Western Blot Analysis of Lung Proteins
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Lung tissues were lysed in precooled RIPA cleavage buffer (Beyotime Biotechnology) for 4 h. Protein concentration was determined using a bicinchoninic acid (BCA) kit (Beyotime Biotechnology). Briefly, 60 μg of total protein was separated via 12% polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane. The membrane was blocked with 5% skim milk for 1 h at room temperature and incubated with primary antibodies (1:1,000) for 6–8 h at 4 °C. After washing with Tris-buffered saline/0.1% Tween 20, the membrane was incubated with the secondary antibodies (1:5,000) for 2 h at 37 °C. Bands were detected using chemiluminescent horseradish peroxidase substrate and quantitatively analysed using Quantum 5.2 software (Bio-Rad). The relative level of immune response was assessed as a grey value and standardised against the reference protein (glyceraldehyde 3-phosphate dehydrogenase) using ImageJ software.
3
Western Blot Analysis of STIL Protein
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The cell lysates were prepared using RIPA cleavage buffer and phenylmethanesulfonylfluoride fluoride (Beyotime Biotechnology, China), to extract the proteins from the cells after full cleavage. The protein was fractionated by SDS-polyacrylamide gel electrophoresis, electroblotted on 0.45 μm polyvinylidene difluoride membranes (Millipore, USA), blocked with 5 % skimmed milk powder at 37 °C for 1 h, washed three times with 1 × PBS-T (1000 mL 1×PBS + 0.5 mL Tween-20), and incubated with the primary antibody (diluted 1:1000) overnight at 4 °C. After three washes, a secondary antibody (diluted 1:10,000) was added to the membrane and incubated for 2 h, and the membrane was washed five times. Finally, an ECL kit (Beyotime Biotechnology, China) was used for detection. Anti-STIL and anti-GAPDH antibodies were purchased from Proteintech. A goat anti-mouse IgG (H + L) -HRP secondary antibody was purchased from Jackson ImmunoResearch. Each experiment was repeated three times.
4
Western Blot Protein Analysis Protocol
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Cell and tissue samples were cleaved in a cold RIPA cleavage buffer (Beyotime, Shanghai, China). Determination of the protein concentration was achieved using a BCA Analytical Kit (Beyotime, Shanghai, China). The proteins were layered by electrophoresis through 10% SDS-PAGE gel (Beyotime, Shanghai, China) and then transferred to 0.22 μm PVDF membranes. The protein-transferred PVDF membrane was blocked with 5% skimmed milk powder for 2 h and subsequently washed three times with TBST, each for 5 minutes, before being incubated with the primary antibody overnight at 4 °C. The primary antibody was recycled, the membrane was washed with TBST three times (each for 5–10 minutes), and then, the secondary antibody was applied for 1 h at room temperature. After three washes with TBST, the ECL (Beyotime, Shanghai, China) was used to visualize the PVDF membrane. For quantification, the optical density of individual bands was analyzed by ImageJ software (V1.8.0, NIH, USA), and the values were normalized to GAPDH (1:5000, Proteintech, Chicago, USA). Full Western blot images can be found in Supplementary File S1 .
5
Protein Expression and Regulation Analysis
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Cells were collected and disrupted with RIPA cleavage buffer (Beyotime, P0013B, China). The lysates were collected after centrifugation and the protein concentration was quantified with BCA kit (Beyotime, P0010S). 50 mg of proteins were added to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE, Beyotime, P0015A) and then were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). These membranes were blocked in 5% non‐fat milk for 1 hours at room temperature and then were treated as the antibody protocol described overnight at 4°C. The following antibodies purchased from Abcam (UK) were used: anti‐Adiponectin (ab181281), anti‐Bcl‐2 (ab32124), anti‐Bax (ab32503), anti‐cl‐Caspase‐3 (ab214430), anti‐LC3II/I (ab192890), anti‐Beclin1 (ab207612), anti‐SQSTM1/p62 (ab207305); anti‐PI3K (ab278545), anti‐phosphor‐Akt (ab38449), anti‐mTOR (ab134903), anti‐NF‐κB (ab16502) and anti‐GAPDH (ab181602). Moreover, the respective Goat anti‐rabbit IgG secondary antibody (Abcam, ab205718) was used to incubate these membranes according to the protocol. The protein bands were quantified with ECL chemiluminescence fluid (Servicebio, G2014, Wuhan, China) and were analysed by Image J software.
6
Protein Extraction and Western Blot Analysis
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At 48 h after transfection, total proteins were extracted with RIPA cleavage buffer (Beyotime Biotechnology, Shanghai, China). After adding 10 μL of the sample buffer to boil for 10 min at 95 °C, SDS-PAGE was achieved at 100 V. After electrophoresis, the protein was transferred to NC membrane at 100 mA for 120 min, sealed with 5% skimmed milk powder for 60 min, and incubated overnight at 4 °C. After the rst antibody was incubated, the membrane was shaken and washed with 1 × TBST solution (Solarbio, Beijing, China) at room temperature, 5min × 3 times, and hybridized with horseradish peroxidase-labeled goat anti-rabbit IgG, at room temperature for 120 min. At 20min intervals, the photoluminescence reaction was performed using the ECL kit (Solarbio, Beijing, China), and protein imprinting was observed. Antibodies are shown in Table 2. The experiment was repeated thrice.
7
Western Blot Protein Analysis Protocol
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At 48 h after transfection, total proteins were extracted with RIPA cleavage buffer (Beyotime Biotechnology, Shanghai, China). After adding 10 μL of the sample buffer to boil for 10 min at 95 °C, SDS-PAGE was achieved at 100 V. After electrophoresis, the protein was transferred to NC membrane at 100 mA for 120 min, sealed with 5% skimmed milk powder for 60 min, and incubated overnight at 4 °C.
After the rst antibody was incubated, the membrane was shaken and washed with 1 × TBST solution (Solarbio, Beijing, China) at room temperature, 5 min × 3 times, and hybridized with horseradish peroxidase-labeled goat anti-rabbit IgG, at room temperature for 120 min. At 20 min intervals, the photoluminescence reaction was performed using the ECL kit (Solarbio, Beijing, China), and protein imprinting was observed. Antibodies are shown in Table 2. The experiment was repeated thrice.
After the rst antibody was incubated, the membrane was shaken and washed with 1 × TBST solution (Solarbio, Beijing, China) at room temperature, 5 min × 3 times, and hybridized with horseradish peroxidase-labeled goat anti-rabbit IgG, at room temperature for 120 min. At 20 min intervals, the photoluminescence reaction was performed using the ECL kit (Solarbio, Beijing, China), and protein imprinting was observed. Antibodies are shown in Table 2. The experiment was repeated thrice.
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