Cells or embryos were lysed in radioimmunoprecipitation assay buffer (P0013B; Beyotime) containing protease inhibitors (P1010; Beyotime). Protein concentrations were determined with the BCA protein assay kit (Thermo). Protein was separated via 8% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and, afterwards, transferred to a polyvinylidene difluoride membrane. After blocking by 5% non-fat milk for 1 hr, primary antibodies were incubated overnight at 4℃. The membrane was washed with tris-buffered saline with Tween-20 and incubated with secondary antibodies for 0.5 hr at room temperature. Bands were detected with the Immobilon ECL Ultra Western HRP Substrate (WBULS0500; Sigma) and band intensity was analyzed by ImageJ software. Antibodies: c-ABL (1:1000, A0282; Abclonal), NOTCH1 (1:1000, 3608 s; CST), and GAPDH (1:1000, ab8245; Abcam). Goat anti-rabbit IgG H and L (HRP) (1:5000, ab6721; Abcam), goat anti-mouse IgG H and L (HRP) (1:5000, ab205719; Abcam).
P1010
Manufactured by Beyotime
Sourced in China
The P1010 is a precision laboratory equipment designed for various scientific and research applications. It is a versatile device that can be used for a range of tasks, though a more detailed description of its core function is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab205719, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Immobilon ecl ultra western hrp substrate, by Merck Group (1 mentions)
Ab8245, by Abcam (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Irisin, by Phoenix Pharmaceuticals (1 mentions)
Ab199426, by Abcam (1 mentions)
P1050, by Beyotime (1 mentions)
P1051, by Beyotime (1 mentions)
P0018, by Beyotime (1 mentions)
Amersham imager 600, by Cytiva (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Bicinchoninic acid (bca), by Beyotime (1 mentions)
Sc 56070, by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Ac038, by ABclonal (1 mentions)
Ab47677, by Abcam (1 mentions)
P0010, by Beyotime (1 mentions)
9 protocols using p1010
1
Western Blot Analysis of Protein Expression
2
Protein Extraction and Immunoprecipitation
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Cultured CFs were lysed in SDS lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% SDS) containing protease inhibitors (Beyotime, P1010). The lysates were denatured by heating for 5 min at 95 °C, diluted 10-fold by lysis buffer, and centrifuged at 20,000×g at 4 °C for 30 min. The supernatant was immunoprecipitated with the indicated antibodies, followed by a Western blot.
3
Quantifying Irisin and Myostatin in Mouse Samples
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After exercise, the mice were anesthetized by pentobarbital (1.5%, 40 mg/kg), the blood was extracted from the heart and then harvested into EDTA-containing tubes and centrifuged at 3000 rpm for 15 min at 4 ℃, and finally, the plasma was collected [11 (link)]. Quadriceps were harvested and then rinsed with pre-cooled PBS at 1:9 weight-volume ratio and then further fully ground with protease inhibitor (Beyotime; P1010; 1:100). Finally, the homogenate was centrifuged at 5000 g for 10 min, and the supernatant was obtained to acquire the quadriceps homogenate. The level of irisin and myostatin in plasma/muscle homogenate was quantified using an ELISA kit according to the protocol of the manufacturer (irisin, Phoenix Pharmaceuticals, Burlingame, CA, USA, NO. EK-067-29/ myostatin, Jianglai, Zhejiang, China, JL50880).
4
Western Blot Analysis of TIAM2 and EGFR Signaling
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The WB was performed as described before [86 (link)]. Proteins of target cells were extracted using a lysate buffer (#87787, Thermo Fisher) containing a protease inhibitor (#P1010, Beyotime). After the protein concentration was determined by BCA assay (#E112-01, Vazyme), 40 μg was used for WB analysis. We purchased the rabbit antibody anti-human TIAM2 (#ab199426, Abcam), mouse monoclonal antibody anti-human β-actin (#AF7018, Affinity), rabbit antibody anti-human p-EGFR (Tyr 1068) (#AF3045, Affinity), rabbit antibody anti-human EGFR (#AF6043, Affinity), rabbit antibody anti-human AKT1/2/3 (Ser473) (#AF0016, Affinity), rabbit antibody anti-human AKT (#AF6261, Affinity), goat anti-rabbit antibody (#S0001, Affinity), and goat anti-mouse antibody (#A21010, Affinity).
5
Aβ Pathology Detection in Mice
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For detecting the pathological soluble or insoluble Aβ in mice, sequential extractions were performed as follows: the half of right hippocampus and cortex from three groups were homogenized in TBS solution containing 5 mM EDTA, phosphatase inhibitor (P1050, Beyotime, Shanghai, China), and protease inhibitor cocktail (P1010, Beyotime) followed by centrifugation at 25,000g at 4°C for 1 h. The supernatant was collected as a TBS fraction and the resulting pellet was solubilized in 70% formic acid (FA), sonicated until the lysis buffer was clear, followed by centrifugation at 25,000g at 4°C for 1 h. The supernatant was collected as the FA fraction and neutralized (1:20) in a neutralization buffer (1 M tris base, 0.5 M Na2HPO4, 0.05% NaN3). Human Aβ40 and Aβ42 were determined by ELISA kits (E-EL-H0452c and E-EL-H0453c, Elabscience, Wuhan, China) in accordance with the manufacturer’s instructions. In addition, the levels of IL-1β (88-7013-88, Invitrogen), IL-6 (EK206HS-96, Multi sciences, HangZhou, China), and TNF-α (88-7324-88, Invitrogen) in TBS fraction were also determined by ELISA.
6
Protein Extraction and Western Blot Analysis
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The cell culture supernatants were removed, washed with PBS, and proteins were extracted by RIPA lysis buffer (R0010, Solarbio, China) with protease inhibitors (P1010, Beyotime, China) and phosphatase inhibitors (P1051, Beyotime, China). The cell lysis mixture was transferred into an EP tube, centrifuged (4°C, 12,000 × g, 10 min), and the supernatants were collected. The separation gel with appropriate concentration was prepared according to the molecular weight of the target protein. The electrophoresis conditions were 80 v 30 min and then 120V until bromophenol blue ran out of the separating gel. Semi-dry transfer conditions: 15 v, 1 h (time adjusted according to protein molecular weight). The membranes were blocked with 5% skim milk or 5% BSA at room temperature for 1 h. After TBST (10 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.2-7.6) washing for four times, the primary antibodies were incubated overnight at 4°C. Remove the primary antibody, wash with TBST for four times, and incubate with the horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. After washing for four times, add color-developing solution (P0018, Beyotime, China) for exposure (Amersham Imager 600).
7
Protein Extraction and Western Blot Analysis
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Fresh tissues (30 mg) were harvested and the proteins were extracted using the RIPA buffer (P0013B, Beyotime Biotechnology) with 1 mM PMSF (ST506, Beyotime Biotechnology) and 1X protease inhibitor cocktail (P1010, Beyotime Biotechnology). The extracted proteins were quantified by BCA (P0010, Beyotime Biotechnology). The primary antibodies listed below were treated with the PVDF membrane at 4°C overnight: anti‐human CASP8 (1:1000 dilution; sc‐56070, Santa Cruz Biotechnology) and anti‐GAPDH (1:1000 dilution; 5174, CST). The membrane was washed with 0.5% TBST thricely for 5 min. Next, the membrane was exposed to secondary antibodies conjugated with HRP (1:5000 dilutions; ZB2305, CST) for 2 hrs. at 25°C. After chemiluminescence, the images were statistically analyzed under the ImageJ software.
8
HVEM Protein Expression Analysis
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Tissues or cells were homogenized in RIPA lysis buffer that contained 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS with freshly added protease inhibitors (P1010, Beyotime Biotechnology, Nantong, China) and phosphatase inhibitor (P1097, Beyotime Biotechnology, Nantong, China). The extract was centrifuged at 12000 ×g for 10 min at 4°C. Supernatants were collected and analyzed with bicinchoninic acid (P0010, Beyotime Biotechnology, Nantong, China) to quantify protein concentration. Proteins were resolved by SDS-PAGE and transferred to PVDF membranes. The rabbit polyclonal Ab against HVEM (Ab47677, Abcam, Cambridge, UK) and β-Actin (Ac038, Abclonal, Wuhan, China). Band intensities were quantified with ImageJ software.
9
Exhaustive Exercise Induces Muscle Fatigue
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The mice were subjected to a single treadmill exhaustive exercise to detect the muscle fatigue strength according to a previous protocol (14) at a speed of 14m/min on a 5° slope by using a Mouse treadmill machine (Anhui Zhenghua Biological Instruments, ZH-PT) at the end of 4 weeks of hypoxic exposure.
ELISA for determining irisin and myostatin concentration in plasma/muscle homogenate Blood samples of anesthetized mice were harvested into EDTA-containing tubes, immediately after the exercise programme, then centrifuged at 3000 rpm for 15 min at 4 ℃, the plasma was collected (15) . Quadriceps were harvested and then rinsed with pre-cooled PBS at 1:9 weight-volume ratio and then further fully grinded with protease inhibitor (Beyotime; P1010; 1:100). Finally, the homogenate was centrifuged at 5000 g for 10 min, and the supernatant was obtained to get the quadriceps homogenate.
The level of irisin and myostatin in plasma/muscle homogenate were quanti ed using an ELISA kit according to the protocol of the manufacturer (irisin, Phoenix Pharmaceuticals, NO. EK-067-29/ myostatin, Jianglai, JL50880).
ELISA for determining irisin and myostatin concentration in plasma/muscle homogenate Blood samples of anesthetized mice were harvested into EDTA-containing tubes, immediately after the exercise programme, then centrifuged at 3000 rpm for 15 min at 4 ℃, the plasma was collected (15) . Quadriceps were harvested and then rinsed with pre-cooled PBS at 1:9 weight-volume ratio and then further fully grinded with protease inhibitor (Beyotime; P1010; 1:100). Finally, the homogenate was centrifuged at 5000 g for 10 min, and the supernatant was obtained to get the quadriceps homogenate.
The level of irisin and myostatin in plasma/muscle homogenate were quanti ed using an ELISA kit according to the protocol of the manufacturer (irisin, Phoenix Pharmaceuticals, NO. EK-067-29/ myostatin, Jianglai, JL50880).
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