Cell pellets were mixed in 200 μl of lysis buffer (2% SDS, 150mM NaCl, 50mM Tris (pH 8.5), proteinase inhibitor mix (Roche, South San Francisco, CA, USA), 5mM DTT, and incubated on ice for 10 min, followed by incubation at 60°C for 45 min. Samples were allowed to cool to room temperature and incubated for 45 min with iodoacetamide to a final concentration of 14 mM. Samples were mixed with 3 parts ice-cold methanol, 1 part chloroform and 2.5 parts H2O, and centrifuged at 4000 g for 10 min. Following removal of the top layer, 3 parts of ice-cold methanol were added, followed by centrifugation at 4000 g for 5 min. Following removal of the top layer, the samples were mixed with 3 parts of ice-cold acetone, vortexed, and centrifuged at 4000 g for 5 min. The pellet was then washed one more time in 2 mL ice-cold acetone, and stored at (−80°C) prior to chemical hydrolysis. The resulting protein pellet was resuspended in 6 N HCl/acetic acid (50:50, 100 μL) and was heated at 95°C for one hour, using a slightly modified version of a reported protocol for protein hydrolysis (Tsugita and Scheffler, 1982 (link)). The resulting aqueous solution was diluted into 40% acetonitrile/40% methanol/20% water and analyzed by LC-MS.
Proteinase inhibitor mix
Manufactured by Roche
Sourced in United States
Proteinase inhibitor mix is a laboratory product designed to inhibit the activity of various proteolytic enzymes, or proteases, which can degrade proteins. It is commonly used in protein extraction and purification workflows to prevent unwanted protein degradation during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico kit, by Thermo Fisher Scientific (1 mentions)
Ecl plus kit, by Cytiva (1 mentions)
Phosphatase inhibitor cocktail 2, by Merck Group (1 mentions)
Protein g beads, by Merck Group (1 mentions)
Pcmv6 hsix1 myc ddk, by OriGene (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Anti flag m2 affinity gel, by Merck Group (1 mentions)
Pparγ, by Santa Cruz Biotechnology (1 mentions)
Cpt1b, by Proteintech (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Phosstop, by Roche (1 mentions)
Pparα, by Abcam (1 mentions)
6 protocols using proteinase inhibitor mix
1
Protein Extraction and Hydrolysis Protocol
2
Western Blot of Plasma Proteins
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Tissue samples were mixed with RIPA buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS), supplemented with 2 mM phenylmethanesulfonyl fluoride, a proteinase inhibitor mix (Roche Applied Science) and phosphatase inhibitor Cocktail 2 (Sigma-Aldrich). Tissue samples were stroked in a Dounce homogenizer, and the homogenates were cleared by centrifugation at 12,000 x g for 15 min at 4°C. Protein concentrations in all samples were measured by the BCA assay kit. Plasma samples were used without further processing (20 μl per sample). Each sample was mixed with 4x loading dye, heated for 5 min at 95°C and then resolved by SDS-PAGE (8-12.5%). Notably, 7.5 μl of original plasma per sample was analyzed. The proteins were transferred to polyvinylidene fluoride membrane, probed with specific antibodies and detected using the ECL Plus Kit (Amersham) or the SuperSignal West Pico Kit (Thermo Scientific).
3
Jurkat Cell Lysis and Western Blot
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1 × 106 Jurkat cells were lysed in NP-40 buffer [1%NP-40, 50 mM Tris-HCl pH 7,6, 150 mM NaCl, 2 mM EDTA, supplemented with proteinase inhibitor mix (Roche), pH 7.5] and total protein concentration was determined using 660 nm Protein Assay (Pierce, Waltham, MA, USA). Total protein amount was adjusted and cell lysates were transferred to SDS-PAGE gels and Western blot analysis was performed using indicated antibodies.
4
Investigating PEPD-mediated ErbB receptor interactions
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PEPD was incubated with ErbB2/ECD-Fc, ErbB3/ECD-Fc, ErbB4/ECD-Fc or Fc in binding buffer for 2 h at 37 °C, followed by pull down with protein G-sepharose beads. The immunoprecipitates were washed with IP washing buffer and analyzed by western blotting. In experiments using whole-cell lysates, cells were lysed in M-PER buffer supplemented with a proteinase inhibitor mix (Roche Applied Science, Indianapolis, IN, USA), and the lysates were incubated with a specific antibody overnight at 4 °C, followed by pull down with protein G-agarose. The immunoprecipitates were washed with IP washing buffer and analyzed by western blotting. To measure the effect of PEPD on PTEN tyrosine phosphorylation, cells were pretreated with 30 μM pervanadate for 10 min to inhibit relevant tyrosine phosphatase25 (link), 30 (link) and then treated with PEPD or vehicle, followed by preparation of cell lysates for analysis. More detail about immmunoprecipitation and preparation of pervanadate is provided in the Supplementary Information .
5
Immunoprecipitation of Transcription Factors
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HEK293 cells were transfected using Lipofectamine 2000 (Invitrogen) with the following plasmids: pCMV6-hSix1:myc:DDK from Origene (RC203465); pcDNA3.1-Cebpa:HA, a kind gift from Dr. D. Tenen; pcDNA3.1-mCebpb from Addgene (12557); pLenti-EF1ap-mEbf2:tGFP (Origene). Three days after transfection, cells were harvested and lysates were prepared as for Western blots except that the lysis buffer contained 50 mM Tris-HCl pH7.4, 150 mM NaCl, 1 mM EDTA, 0.1% NP40, and proteinase inhibitor mix (Roche, 11 873 580 001). 500 μg of total proteins were pre-cleared with 10 μl protein G beads (Sigma, P3296) and incubated overnight with 10 μl anti-FLAG M2 affinity gel at 4°C on a rotator (Sigma, A2220). The gel was washed twice each with lysis buffer, RIPA buffer containing 0.1% SDS, RIPA buffer containing 0.1% SDS and 1% NP-40. Precipitated proteins were eluted using SDS loading buffer (95°C, 5 min) and separated on PAGE gels for Western analysis.
6
Western Blotting of Muscle Proteins
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The muscle tissue (~100 mg) was homogenized with a steel bead in 1 ml of cold RIPA buffer containing 1X Proteinase Inhibitor Mix (complete™ Protease Inhibitor Cocktail, Roche, Cat. no.11 697 498 001), 1X PhosStop (Roche, Mannheim Germany, Cat. no.04 906 845 001) using a TissueLyser II instrument (Qiagen) set at 30 strokes/s for 2–4 min. Based on protein quantification results, all samples were adjusted to the final concentration of 2 μg/ul and heat-denatured for 5 min at 99 °C in 2X Laemmli buffer. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated in Odyssey blocking solution for 1 h. Total proteins were detected by probing the membranes with appropriate primary antibodies overnight at 4 °C. The following antibodies were used: Chkα (1:1000, Abcam Cat#ab88053), Pparα (1:1000, Abcam, Cat#Ab24509), Pparb (1:1000, Biorad, Cat#AHP1272), Cpt1b (1:1000, Proteintech®, Cat#22170-1-AP), Chkβ (1:250, Santa Cruz, Cat#398957), GAPDH (1:1000, Cell signaling, Cat#2118), Pparγ (1:500, Santa Cruz, Cat# sc-7273). Proteins were visualized with goat anti-rabbit IRDye-800- or −680-secondary antibodies (1:20,000, LI-COR Biosciences, Cat#926-32211 and Cat#926-68071) or anti-mouse m-IgGκ BP-CFL 790 (Santa Cruz, Cat. no.sc-516181) using an Odyssey imaging system and band density were evaluated using FIJI (NIH).
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