The total proteins were extracted from tissues and cells using RIPA (#P0013B, Beyotime) supplemented with protease and phosphatase inhibitors (#P1005, Beyotime). The concentration of extracted proteins was determined using the BCA method (#P0010, Beyotime). Extracted proteins were separated by 10% SDS-PAGE and transferred to poly (vinylidene fluoride) (PVDF) membranes (#IPVH00010, Millipore). The membranes were blocked using the blocking buffer (5% evaporated milk) and incubated using the indicated primary antibody overnight at 4°C. The membranes were incubated using horseradish peroxidase (HRP) conjugated secondary antibodies for 1 h at the room temperature and washed using tris-buffered saline and Tween 20 (TBST). The western blots were developed using enhanced chemiluminescence (#E412-02, Vazyme). The antibodies used are listed in Supplementary Table 5 .
P1005
Manufactured by Beyotime
Sourced in China
The P1005 is a laboratory equipment designed for precise temperature control. It features a digital display and adjustable temperature settings to maintain the desired temperature within the specified range.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (6 mentions)
St506, by Beyotime (5 mentions)
Ipvh00010, by Merck Group (4 mentions)
P0013, by Beyotime (4 mentions)
Sc 3879, by Santa Cruz Biotechnology (3 mentions)
Sc 33652, by Santa Cruz Biotechnology (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
P1081, by Beyotime (3 mentions)
Bca kit, by Beyotime (2 mentions)
Ffp24, by Beyotime (2 mentions)
P0012, by Beyotime (2 mentions)
St041, by Beyotime (2 mentions)
P2051, by Beyotime (2 mentions)
St505, by Beyotime (2 mentions)
Ecl substrate reagent kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
32 protocols using p1005
1
Protein Extraction and Western Blotting
2
Western Blot Analysis of Cellular Signaling
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Cells and kidney tissues were homogenized in RIPA lysis buffer containing a protease inhibitor cocktail (P1005, Beyotime). Subsequently, the protein concentrations were determined using the BCA kit (P0012S, Beyotime). The protein extracts were separated on 7.5% or 12.5% SDS-PAGE gels (120 V, 90 min) and further electro-transferred to FVDF membranes (80 V, 90 min). After being blocked with 5% bovine serum albumin, the membranes were incubated at 4 °C overnight with the appropriate primary antibodies (Camkk2, 11549-1-AP, Proteintech; p-Camkk2, 12818, CST; AMPK, 2532, CST; p-AMPK, 2535, CST; GPX4, ab125066, Abcam; SLC7A11, 11549-1-AP, Proteintech;). Following this, the membranes were washed with TBST for 5 × 5 min and subsequently incubated with HRP-conjugated secondary antibody (LF102, Epizyme, Shanghai) for 1 h at room temperature. Finally, the bands on the membranes were visualized and analyzed by electrochemiluminescence. The expression of β-actin (4970, CST) was utilized as a control.
3
Quantification of Protein Expression
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Total protein was extracted using the RIPA buffer (PC101, Epizyme, Inc., Cambridge, MA, USA) containing a protease inhibitor cocktail (P1005, Beyotime, Beijing, China). The BCA method (P0011, Beyotime) was used to detect the protein concentration. Total proteins (20 μg) were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were probed with antibodies (18,309-1-AP for ITGB3, 17,670-1-AP for JAK2, and 66,009-1-Ig for β-actin from Proteintech, China). The protein bands were detected with an Amersham Imager 600 (GE Healthcare, Waukesha, WI, USA).
4
Quantifying Protein Expression in Neurodegenerative Disease
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Protein expression levels of APP, Beclin-1, p62, LC3, Aβ1-42, and β-actin were detected by western blot. After cells were treated for 24 h, they were collected, and total proteins were extracted using RIPA buffer (P0013B, Beyotime) mixed with a protease inhibitor mixture (P1005, Beyotime). Protein concentrations were determined using a BCA assay kit (P0012, Beyotime). Protein samples mixed with loading buffer (P0015, Beyotime) were separated via SDS-PAGE (P0012AC, Beyotime) and transferred to PVDF membranes (FFP24, Beyotime). The membranes were blocked with 5% (w/v) BSA (4240GR025, Biofroxx) for 1 h at room temperature and incubated with primary antibodies (1:1,000 dilution) at 4°C overnight. Subsequently, the membranes were treated with HRP-conjugated goat anti-rabbit antibodies (1:1,000 dilution). Finally, the membranes were visualized with an enhanced chemiluminescence reagent kit (WBKLS0100, Millipore, United States) and photographic imaging equipment (ChemiDoc MP, Bio-Rad, United States). Relative band intensities were quantified using Image J software and normalized to β-actin.
5
Mouse Oocyte Purification Protocol
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Oocyte purification was performed as described previously [69 (link)]. In brief, mouse ovaries were isolated and digested with 90 μl of TrypLE (12,604,021, Thermo, New York, US) at 37 °C for 10 min under constant shock. Digestion was terminated by adding 10 μl foetal bovine serum (C0235, Beyotime, Shanghai, China). After centrifugation at 1000 rpm for 1 min, the supernatant was discarded, and the cells were treated with 80 μl hypotonic buffer (30 mM Tris–HCl, 17 mM trisodium citrate dihydrate, 50 mM sucrose, 5 mM EDTA, 0.5 mM DTT, pH 8.2) containing proteinase inhibitor (1:100, P1005, Beyotime, Shanghai, China) for 30 min. Slides were pretreated with 20 μl of fixation buffer (1% paraformaldehyde, pH 9.2 with 50 mM boric acid) containing 0.15% Triton X-100 (T9284, Sigma, Missouri, USA) applied evenly on slides in advance. Then, 20 μl aliquots of the cell suspension were dripped onto the slides, which were incubated at 37 °C for 4 h in a humidified box. The samples were left to dry at room temperature. Then, immunofluorescence staining was performed according to the protocol described above.
6
RNA Immunoprecipitation of SRSF1 in Mouse Ovaries
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As described previously [70 (link)], RIP was performed using 16.5 dpc mouse ovaries. The ovaries were lysed in cell lysis buffer (P0013, Beyotime, Shanghai, China) containing PMSF (1:100, ST506, Beyotime, Shanghai, China), a proteinase inhibitor cocktail (1:100, P1005, Beyotime, Shanghai, China), DTT (1:50, ST041-2 ml, Beyotime, Shanghai, China), and an RNase inhibitor (1:20, R0102-10 kU, Beyotime, Shanghai, China). After incubation on ice for 20 min, the lysate was added to 20 μl of protein A agarose (P2051-2 ml, Beyotime, Shanghai, China) for preclearing at 4 °C for 1 h. Two micrograms of an SRSF1 antibody (sc-33652, Santa Cruz Biotechnology, California, USA) and a normal mouse IgG (sc-3879, Santa Cruz Biotechnology, California, USA) were added to the lysate, followed by overnight incubation at 4 °C. The next day, 60 μl of protein A agarose was added to the lysate followed by incubation at 4 °C for 4 h. The agarose complexes containing antibodies, target proteins, and RNA were washed for 5 min at 4 °C, which was repeated 3 times. Protein-bound RNA was then extracted using RNAiso Plus and the Direct-zol RNA MicroPrep kit. RIP–qPCR was performed according to the above RT–qPCR protocol.
7
Western Blot Analysis of Osteogenic Markers
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In the presence of a protease inhibitor (Beyotime, China, P1005), the osteogenic hFOB 1.19 cells were lysed using RIPA lysis buffer (Beyotime, China, P0013B), and centrifugation at 4 °C (12,000 rpm) for 15 min collected the supernatant. Protein samples were separated by SDS–PAGE electrophoresis and then transferred to polyvinylidene fluoride (PVDF; Millipore, IPVH20200) membranes. The TBST containing 5% skim milk (Beyotime, China, P0216) was used to block the nonspecific binding site for 1 h, and the antibodies were incubated overnight at 4 °C. After incubation with the antimouse IgG (Jackson, 115-035-003) or antirabbit IgG (Jackson, 111-035-003) at room temperature for 1 h, the signal was detected with an enhanced chemiluminescence reagent (Beyotime, China, P0018FM). Table 2 presents the list of the primary antibodies.
The primary antibody information of this study
| Primary antibody name | Company and catalog | Dilution | Molecular weight (kDa) |
|---|---|---|---|
| RUNX2 | CST, 8486 | 0.736111111 | 55–62 |
| OPN | abcom,ab8448 | 0.736111111 | 66 |
| OCN | Abcam, ab133612 | 0.736111111 | 11 |
| GAPDH | Proteintech 60,004–1-Ig | 5.597222222 | 36 |
8
Western Blotting Quantification Protocol
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Western blotting was performed as previously described [20 ]. Proteins were collected using RIPA buffer (P0013B, Beyotime, China), protease inhibitors (p1005, Beyotime, China), and phenylmethylsulfonyl fluoride (ST505, Beyotime, China), quantified by a protein quantification kit (P0011, Beyotime, China), and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, electrophoresis was conducted to transfer the proteins into a PVDF membrane (160-0184, Bio-Rad, USA). The membrane was blocked by 5% dried skimmed milk powder for 1 h and then incubated with antibodies against Akt (1: 500, 56kDa, ab8805, Abcam, UK), phosphorylated (p)-Akt (1: 1000, 56kDa, ab38449, Abcam, UK), endothelial nitric oxide synthase (eNOS) (1: 1000, 133kDa, ab76198, Abcam, UK), p-eNOS (1: 500, 140kDa, ab76199, Abcam, UK), and GAPDH (1: 1000, 36kDa, ab8245, Abcam, UK). After incubation for 24 h, the membrane was washed by 1% Tris-buffered saline with Tween 20 and incubated with goat anti-rabbit secondary antibody (1: 10 000, ab205718, Abcam, UK) or goat anti-mouse secondary antibody (1: 10000, ab6789, Abcam, UK). An ECL luminescence kit (PE0010, Solarbio, China) and a gel imaging system (FluorChem FC3, Alpha, USA) were used to expose the membrane and visualize the protein bands, respectively. ImageJ2x (Rawak Software, Germany) was used to analyze the results.
9
Liensinine-induced Apoptosis and Autophagy
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Cells were seeded into 6-well plates with 1∗105 cells each well. Liensinine was prepared for different concentrations (0, 2.5, 5, 10, or 20 μM) and added into the wells for 48h. After washing PBS for 3 times, cells were lysed with lysis buffer (P0013 J, Beyotime, China) supplemented with protease inhibitor (P1005, Beyotime, China) and phosphatase inhibitor (P1081, Beyotime, China). After centrifuging at 12000 × g for 15 min at 4°C, the concentration of the supernatant was detected by a BCA kit (23225, Thermo Fisher Scientific, USA). And then, Western blot assay was carried out in a routine process [22 (link)]. The results were visualized using ECL substrate reagent kit (32209, Thermo Fisher Scientific, USA) or detected by exposure to a film. The primary antibody dilution is as follows: cleaved-PARP (1 : 1000), caspase 3 (1 : 1000), cleaved-caspase 3 (1 : 500), cleaved-caspase 9 (1 : 500), BAX (1 : 1000), cytochrome c (1 : 1000), LC3B (1 : 500), SQSTM1 (1 : 1000), mTOR (1 : 1000), phospho-mTOR (1 : 1000), AMPK (1 : 1000), phospho-AMPK (1 : 1000), Beclin1 (1 : 1000), ULK1 (1 : 1000), LAMP2 (1 : 1000), LAMP1 (1 : 1000), and ACTB (1 : 1000). Anti-rabbit IgG was diluted at a ratio of 1 : 2000. Primary antibody was incubated overnight at 4°C, and secondary antibody was incubated for 1 hour.
10
Quantitative Western Blot Analysis
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For total protein extraction, the cells were washed with PBS, lysed in Radio-Immunoprecipitation Assay buffer (R0020, Solarbio) with phosphatase/protease inhibitor (P1005, Beyotime), and measured by ultraviolet spectrophotometry. The lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride membrane. The membranes were blocked with 5% bovine serum albumin and incubated with primary antibodies raised against GAPDH (1:1000; 5174S, Cell Signaling Technology, Danvers, MA, USA), PDK4 (1:500, 12,949-1-AP, Proteintech, Rosemont, IL, USA), p44/42 MAPK (ERK1/2) (1:2000, 4695S, Cell Signaling Technology), and phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:2000, 4370S, Cell Signaling Technology). Goat anti-rabbit antibodies conjugated to horseradish peroxidase were used as secondary antibodies (1:5000; ZB-2301, ZSGB-BIO, Beijing, China), and the proteins were visualized using enhanced chemiluminescence (JIAPENG, JP-K600plus, Shanghai, China). The band intensity was quantified using the ImageJ software (US National Institute of Health, Bethesda, NC, USA). The experiments were performed in triplicate.
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