Total protein in skin flap was collected by RIPA lysate (R0278, Sigma-Aldrich, Missouri, USA) mixed with protease inhibitor (S8830, Sigma-Aldrich, Missouri, USA) in order to determine the protein levels of RIP1, RIP3, MLKL, PGAM5, Drp1, MKP-1, p-ERK1, p-ERK2, ERK1 and ERK2. The concentration of total protein was evaluated by BCA kit (Sigma-Aldrich, Missouri, USA) in the first place to estimate the loading volume of samples onto the SDS-PAGE for separation. Parameter for electrophoresis was 100 V for 2 h. PVDF membrane was used for the transferring of protein separated. Following the blocking of membrane for 60 min, primary antibodies against RIP1 (CST, #3493, 78 kD, 1:1000), RIP3 (Abcam, ab56164, 57 kD, 1:1000), MLKL (CST, #37705, 54 kD, 1:1000), PGAM5 (Abcam, ab126534, 32 kD, 1:1000), Drp1 (Abcam, ab56788, 82 kD, 1:1000), MKP-1 (CST, #3493,78 kD, 1:1000), p-ERK1/2 (Abcam, ab50011,42–44 kD, 1:1000), ERK1/2 (Abcam, ab17942, 42–44 kD), GAPDH (Abcam, ab8245, 36kD, 1:2000) were incubated on the membrane at 4 °C overnight. Next day, after the washing of membrane for 3 times, primary antibodies were probed by Goat anti-rabbit secondary antibody (Abcam, ab205718, 1:2000) and developed by ECL (#6883, SignalFire™ ECL Reagent) after washing by PBST (PBS with 0.1% Tween).
S8830
Manufactured by Merck Group
Sourced in United States
The S8830 is a laboratory equipment product from Merck Group. It is a highly precise and versatile instrument designed for a range of scientific applications. The core function of the S8830 is to perform accurate measurements and analysis. However, a detailed unbiased and factual description of the product's features and capabilities is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Ab205718, by Abcam (2 mentions)
Ripa lysate, by Merck Group (2 mentions)
Ab56788, by Abcam (1 mentions)
Ab50011, by Abcam (1 mentions)
Ab17942, by Abcam (1 mentions)
Ab56164, by Abcam (1 mentions)
Ab126534, by Abcam (1 mentions)
Bca kit, by Merck Group (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Ab8245, by Abcam (1 mentions)
Ni nta agarose beads, by Thermo Fisher Scientific (1 mentions)
Nb7539, by Novus Biologicals (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Np40s, by Merck Group (1 mentions)
S6508, by Merck Group (1 mentions)
Ecl blotting substrate, by Advansta (1 mentions)
Ab15348, by Abcam (1 mentions)
P8215, by Merck Group (1 mentions)
Tris buffered saline and tween 20 tbst, by Merck Group (1 mentions)
Image lab software version 3, by Bio-Rad (1 mentions)
14 protocols using s8830
1
Protein Expression Analysis of Skin Flap
2
Tissue Extraction and Protein Quantification
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The uterine tissue (30 mg; endometrium and myometrium separate) was homogenized on ice in RIPA lysis buffer (5 mM EDTA, 150 mM NaCl, 50 mM TRIS, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, and a protease inhibitor from Sigma-Aldrich, S8830, pH 7.4). The obtained lysate was centrifuged at 10,000× g for 20 min at 4 °C, and the supernatant was transferred to a fresh tube and sonicated. The protein concentration was estimated according to Bradford’s method. The lysate was stored at −80 °C until further analysis.
3
Western Blot Analysis of ATG5 and LC3
4
Purification of His-Tagged Proteins from E. coli
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The plasmids expressing proteins with an N-terminal hexahistidine tag were transformed in E. coli BL21 strain. Cells were grown in Terrific Broth (TB) at 37 oC. The expression was induced at 30 oC or 16 oC by addition of isopropyl β-D-1-thio-galactopyranoside (IPTG) to a final concentration of 1 mM at OD(600) = 0.6. The cells were harvested after 16 h of expression and lysed by sonication in lysis buffer (50 mM PBS with 150 mM NaCl at pH 7.4, 5 mg/ml DNAse, 5 mM MgCl2, and 1 mM phenylmethylsulfonyl fluoride (PMSF), and a cocktail of protease inhibitors (Sigma Aldrich; S8830)). After lysis, the mixture was incubated on ice for 2 h for DNA digestion. The insoluble material was removed by centrifugation and the supernatant was incubated overnight with Ni-NTA agarose beads (Thermo Fisher) at 4 oC in a rotator-mixer. The protein-loaded Ni-NTA column was washed twice with 20 column volumes of N1 buffer (50 mM PBS, 300 mM NaCl, 30 mM imidazole, pH 7.4) and twice N2 buffer (50 mM PBS, 150 mM NaCl, 10 mM imidazole, pH 7.4). The bound protein was eluted with N3 buffer (150 mM PBS pH 7.4, 300 mM imidazole). The protein fractions were eventually dialyzed with cassette Slide-A-Lyzer Dialysis Cassettes (Thermo Fisher) against 50 mM PBS, 150 mM NaCl pH 7.4.
5
Western Blot Analysis of OPA1 Protein
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Cells were harvested and washed with PBS, and the cell pellet was resuspended in lysis buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 4% glycerol, 1% Triton X-100, 0.1% sodium deoxycholate, 0.05% SDS) supplemented with protease inhibitors (S8830, Sigma-Aldrich). After 30 min incubation on ice, the supernatant was cleared from insoluble debris by centrifugation (30 min, 15,000g, 4°C). Forty micrograms of total protein lysate was loaded per lane, separated by 12% SDS-PAGE, and blotted onto a polyvinylidene fluoride (PVDF) membrane. After being blocked with 5% BSA/TBST for 1 h at room temperature, the membrane was incubated overnight at 4°C with an OPA1-specific antibody (1:1,000 diluted, mouse anti-OPA1 clone 18, cat. no. 612,607; BD Biosciences, Heidelberg, Germany) in blocking buffer. As loading control, a mouse anti-GAPDH antibody (Merck, Chemicon, MAB374) was used. A peroxidase-conjugated goat anti-mouse antibody (NB7539, Novus) was used as secondary antibody, followed by enhanced chemiluminescence (ECL) detection. Relative protein levels were calculated based on band intensity quantifications using ImageLab software (Bio-Rad).
6
Western Blot Analysis of PBMC Proteins
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A total of 5 × 106 PBMCs were washed twice with 1X PBS and centrifuged at 1500 rpm for 5 min. Cells were lysed in a 1X RIPA buffer containing 150 mM NaCl, 1% NP-40 (Sigma Aldrich; NP40S), 50 mM Tris-HCl, pH 8, 0.5% deoxycholic acid (Sigma Aldrich, D6750), 0.1% SDS (Sigma Aldrich, 71736), protease (Sigma Aldrich; S8830) and phosphatase inhibitors (Sodium Orthovanadate; Sigma Aldrich; S6508) (Sodium Fluoride; Sigma Aldrich; S7920). Then, 15 µg of protein lysates was subjected to electrophoresis on 4–20% SDS-PAGE gradient gels (NuSep, Germantown, MD, USA; NN12-420), according to the manufacturer’s instructions. Then, the gels were transferred to nitrocellulose membranes (Bio-Rad, 162-0115) for 2 h in Tris-glycine buffer. The membranes were blocked in PBS-0.1% Tween 20 solution containing 3% BSA, probed with specific antibodies, and developed using ECL Blotting Substrate (Advansta, San Josè, CA, USA; K-12045-D20).
7
ACE2 Protein Extraction and Quantification
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Gene-edited A549-ACE or Calu-3 cells seeded in six-well plate 24 h prior to experiment were chilled on ice for 10 min, and labeled with 2.5 mg/ml biotin (Thermo Fisher #21331) in PBS for 30 min on ice. Cells were quenched with 100 mM glycine in PBS three times, 10 min each. After washing with PBS, cells were lysed in RIPA buffer (Cell Signaling #9806S) with a cocktail of protease inhibitors (Sigma-Aldrich # S8830), and immunoprecipitated with Streptavidin agarose beads overnight at 4 °C. Beads were then washed three times with RIPA buffer, and eluted into 5× loading buffer (Beyotime #P0015L) at 95 °C for 10 min. After spinning at maximum speed for 10 min, the supernatants were harvested for western blotting using rabbit anti-ACE2 (Abcam #ab15348, 1:1000) as described above, and analyzed with the Odyssey CLx Imaging System and Image Studio 4.0 software. The un-immnoprecipitated lysates were used as a loading control.
8
Mushroom Protein Extraction and Purification
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Frozen mushroom pulp was defrosted at 4 °C and suspended in a 125 mM sodium citrate buffer, pH 5.3, containing 25 mM sodium l -ascorbate, 20 mM l -lysine, 50 mM l -proline. Again, PSMF and benzamidine hydrochloride pre-solved in DMSO were added (final concentration 1 mM), as well as two protease inhibitor cocktails (P8215 0.1% (v/v) and S8830 1 tab/L). The suspension was stirred for 15 min and subsequently centrifuged (14,500 rpm at 4 °C over 10 min). The pellet and the highly viscose triton-phase, covering the pellet, were discarded. The supernatant was adjusted to 20% (113 g/L) ammonium sulfate saturation and centrifuged (14,500 rpm at 4 °C over 10 min). The occurring pellet was discarded and PEG-4000 was dissolved to a concentration of 15% (m/v) in the supernatant at 4 °C. After further centrifugation (14,500 rpm, 4 °C, 10 min) the generated PEG phase (upper phase) containing the non-target proteins was discarded. This step was followed by the subsequent addition of another 5% (m/v) of PEG-4000 and centrifugation. This last step was repeated twice. During the extraction/purification process every 45 min a protease inhibitor mixture containing PMSF, benzamidine hydrochloride and the two cocktails P8215/S8830 (Sigma–Aldrich) pre-dissolved in DMSO was added.
9
Western Blot Protein Analysis Protocol
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RIPA lysate (R0278; Sigma-Aldrich; Merck KGaA) and a protease inhibitor were used to lyse cells (1×106) and extract total protein (S8830; Sigma-Aldrich; Merck KGaA). Each sample's total protein (50 µg per lane) was separated on a 10% SDS-PAGE at 120 V for around 1.5 hours. The protein suspension was mixed with 1X antibody solution, which was then transferred to a PVDF membrane and sealed with 5% skim milk for 1 hour at room temperature. The cell membranes were sealed with skim milk and then treated with a primary antibody overnight at 4°C (Sigma-Aldrich; Merck KGaA). The membranes were incubated with the matching secondary antibodies for 1 hour at room temperature the next day, after being washed with Tris-buffered saline and Tween 20 (TBST; Sigma-Aldrich, St. Louis, MO, USA). To find the proteins, Cell Signaling Technology, Inc.'s SignalFire™ ECL reagent (Catalog #6883) was used. Protein blot results were optical density analyzed and quantified using ImageLabTM software (version 3.0) from Bio-Rad Laboratories Inc.
10
Western Blot Analysis of Protein Lysates
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Cells or xenograft tumors were lysed using RIPA (catalog V900854-100ML, VETEC) with 1 mM PMSF, and protease inhibitors (catalog S8830, Sigma‒Aldrich). Protein concentrations were quantified using the BCA protein test package (catalog P0010, Beyotime). After the proteins were separated, they were transferred to a PVDF membrane (catalog IPVH00010, Millipore), blocked with 5% nonfat milk, and incubated with the corresponding antibodies. On the second day, the bands were incubated with the corresponding secondary antibodies. HRP chemiluminescent substrates were used to visualize the immunoreactive bands (catalog BMU102-CN, Abbkine). The bands were quantified from the western blot results using Image Lab. The antibodies used in this study are listed in Supplementary Table 2 .
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