Tight-junction proteins were assessed for changes to Triton X-100 extractability as described earlier12 (link) with modifications. After 6 days in culture on Transwell-permeable supports, AECs were washed 2 × with ice-cold DPBS. After washing, four wells were combined and cells were scraped 2 × into ice-cold DPBS containing protease inhibitor cocktail with EDTA (Roche). Cells were centrifuged for 8 min at 500 g at 4 °C, resuspended in DPBS with protease inhibitor cocktail (Roche) containing 0.1% (v/v) Triton X-100 and incubated for 30 min at 4 °C. Next, cells were centrifuged at 100,000 g for 30 min at 4 °C, to separate the lysate into Triton-soluble (supernatant) and -insoluble (pellet) fractions. The samples were equivalently diluted in SDS–PAGE sample buffer, heated for 10 min at 70 °C, then analysed by SDS–PAGE and immunoblotting as described above.
Protease inhibitor cocktail with edta
Manufactured by Roche
Sourced in United States
Protease inhibitor cocktail with EDTA is a laboratory reagent designed to inhibit a broad range of proteolytic enzymes, also known as proteases. The inclusion of EDTA, a chelating agent, further enhances the inhibitory properties of the cocktail. This product is intended for use in various research and analytical applications where the preservation of protein samples is crucial.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (1 mentions)
Optima l 90k ultracentrifuge, by Beckman Coulter (1 mentions)
Bugbuster protein extraction reagent, by Merck Group (1 mentions)
Igepal ca 630, by Merck Group (1 mentions)
Versadoc 4000, by Bio-Rad (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Phosstop, by Roche (1 mentions)
Gfp trap agarose, by Proteintech (1 mentions)
Mouse anti flag m2, by Merck Group (1 mentions)
Mouse anti myc 9e10, by Santa Cruz Biotechnology (1 mentions)
Mouse anti v5 r960 25, by Thermo Fisher Scientific (1 mentions)
Mouse anti tubulin e7, by Developmental Studies Hybridoma Bank (1 mentions)
Rabbit anti ha, by Cell Signaling Technology (1 mentions)
Anti flag m2 affinity gel, by Merck Group (1 mentions)
Rat anti ha 3f10, by Roche (1 mentions)
5 protocols using protease inhibitor cocktail with edta
1
Triton X-100 Extraction of Tight-Junction Proteins
2
Subcellular Fractionation and Protein Analysis
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Cells in 3 confluent 15-cm dishes were washed in ice-cold PBS, scraped in 10 ml SHB and pelleted by centrifugation. Cells were resuspended in 2 ml SHB buffer (0.25 M sucrose, 1 mM EDTA, 10 mM HEPES, pH = 7.4) plus protease inhibitor cocktail with EDTA (Roche) and 1 mM PMSF and disrupted by 30 strokes in a steel dounce homogenizer. The disrupted cells were centrifuged for 12 min at 1400 g to remove nuclei. Supernatants were loaded onto continuous sucrose gradients (percentage iodixanol: 0, 10, 20, 30) and ultracentrifuged in an SW41 rotor at 181,299 x g for 1 h (Optima L-90K Ultracentrifuge, Beckman Coulter). Twenty-two fractions of 420 µl were collected from top to bottom, and 100 µl of each fraction were denatured in SDS buffer for western blot analysis.
3
Leptospira Protein Extraction Protocol
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The L. interrogans serovars Lai, Canicola, Copenhageni, and the non-pathogenic serovar L. biflexa serovar Patoc were grown in liquid EMJH medium and harvested by centrifugation at 18,514 g for 10 mins. Cells were washed twice with 1X PBS pH 7.4 and pellets were resuspended in 5 mL/gram of BugBuster® Protein Extraction Reagent (Sigma-Aldrich, USA) containing “Protease Inhibitor Cocktail with EDTA” (Roche, USA). Cell lysates were incubated on a rotating mixer for 15 minutes at room temperature. Insoluble cell debris was removed by centrifugation at 18,514 g for 20 minutes at 4°C. Supernatant were stored at -20°C until analysis.
4
Western Blotting for Brain and Neuronal Proteins
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Western blotting was performed as previously described12 (link). Brain tissue, primary hippocampal neurons and 293T cells were homogenized in an ice-cold lysis buffer containing 1% IGEPAL CA-630 (Sigma), 0.5% Triton X-100, 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and supplemented with phenylmethanesulfonyl fluoride solution and a protease inhibitor cocktail with EDTA (Roche). Whole-cell lysates were subjected to further mechanical disruption by passage through a 26-gauge syringe 5–6 times. Lyates were cleared by centrifugation at 16,100g for 30 min at 4 °C and the solubilized fraction of protein was used for all biochemical experiments. Lysis buffer used in experiments requiring detection of phospho-tyrosine was additionally supplemented with a phosphatase inhibitor cocktail (PhosSTOP, Roche; 04906845001). Proteins were separated by SDS–PAGE, analysed by immunoblotting with the indicated antibodies and visualized using enhanced chemiluminescence (Pierce Biotechnology) on a Bio-Rad Versadoc 4000 (Bio-Rad Laboratories). Blots were quantified using Image J software. Full-length blots with molecular weight markers, denoting kDa, are shown in Supplementary Fig. 5 .
5
Co-Immunoprecipitation Assay Protocol
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S2 and HEK293T cells were lysed with HEPES lysis buffer (150 mM NaCl, 50 mM HEPES pH 7.5, 0.5% (v/v) Triton X-100 supplemented with protease inhibitor cocktail with EDTA (Roche) and the soluble fraction was obtained by centrifugation (16,000 × g, 20 min). For co-IP experiments cleared lysates were added to 20 μL of ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich) or 15 μL of GFP-Trap agarose (ChromoTek) in 100 μL HEPES lysis buffer. The lysate and the beads were incubated for 1 h at 4 °C and washed four times with HEPES lysis buffer. Input and co-IP samples were analysed by SDS-PAGE and western blot. Primary antibodies were: mouse anti-FLAG (M2), 1/5000 (Sigma-Aldrich F1804); mouse anti-GFP (3E1), 1/1000 (Cancer Research UK); rat anti-HA (3F10), 1/2000 (Roche); mouse anti-Myc (9E10), 1/5000 (Santa Cruz sc:40); mouse anti-Tubulin (E7), 1/5000 (Developmental Studies Hybridoma Bank); mouse anti-V5 (R960-25), 1/5000 (Thermo Fisher), rabbit anti-P-TAZSer89 1/1000 (E1X9C) (Cell Signalling Technologies 59971), rabbit anti-HA 1/1000 (C29F4) (Cell Signalling Technologies 3724). Secondary antibodies were from GE Healthcare (1:5000).
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