Sentences with phrase «elb lysis buffer»

Total proteins were harvested in lysis buffer (20 mM Tris - HCl [pH 8.0], 150 mM NaCl, phosphatase inhibitors [Pierce], protease inhibitors [C complete protease inhibitor cocktail, Roche], and 1 % Triton X-100).

Peripheral blood mononuclear cells were isolated by centrifugation with 96 % Ficoll (BD), followed by erythrocyte lysis with ACK lysis buffer.

At 48 hours, cells were washed twice with PBS and lysed in 1 × passive lysis buffer (Dual - Luciferase Reporter Assay, Promega).

The SVCs were resuspended in erythrocyte lysis buffer and incubated at room temperature for 5 minutes.

Lane 1: HEK cells Lane 2: hfRPE cells Lane 3: hfRetina PVDF membrane RIPA lysis buffer with mixed phosphatase and protease inhibitors

Single animals were placed into 5 µl of worm lysis buffer (10 mM Tris — HCl pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 0.45 % NP - 40, 0.45 % Tween 20, 0.01 % Gelatin and 500 µg / ml fresh proteinase K) in a PCR tube.

Spleens were processed into cell suspensions through a 70 - μm strainer by a syringe plunger in RPMI 1640 with 10 % FBS and then treated with ACK lysis buffer to remove RBCs.

Reactions were terminated with 1 ml of ice - cold PBS, and cells were centrifuged and resuspended in 50 μl of cell lysis buffer containing 1 % NP - 40 and protease inhibitors.

Lane 1: HEK cells Lane 2: hfRPE cells Lane 3: hfRetina Lane 4: Mouse liver Lane 5: Mouse RPE Lane 6: Mouse retina Lane 7: Zebrafish liver Lane 8: Zebrafish retina PVDF membrane RIPA lysis buffer with mixed phosphatase and protease inhibitor cocktail

Beads were washed by centrifugation at 100g, 4 °C for 2 min with lysis buffer containing 2 mM Na3VO4 and boiled with 2 × protein sample buffer at 95 °C for 10 min.

Cells were lysed on ice using lysis buffer (10 mM Tris - HCl pH 7.4, 150 mM NaCl, 1 % Triton X-100, protease inhibitor mix G (Serva) and 2 mM Na3VO4).

Spleens were then treated with ACK lysis buffer to remove RBCs.

Cells were lysed in RIPA lysis buffer.

After extensive washing with immunoprecipitation lysis buffer, the immunoprecipitated proteins were analyzed by immunoblotting using specific antibodies against DDX3 and FLAG epitope (ab1257, Abcam, Cambridge, United Kingdom).

Hippocampal and entorhinal cortex tissue samples were homogenized in 10 volumes of RIPA lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1 % sodium dodecyl sulfate and 0.5 % deoxycholate, and 1 % NP40) containing a cocktail of protease and phosphatase inhibitors (20 mg / ml each of pepstatin A, aprotinin, phosphoramidon, and leupeptin; 0.5 mM 4 -(2 - aminoethyl) benzenesulfonyl fluoride hydrochloride; 1 mM EGTA; 5 mM fenvalerate; and 5 mM cantharidin).

For the coimmunoprecipitation experiments, HuH - 7 cells transfected with pcDNA - SRα / FLAG - Sp 1 were harvested and cell lysates were prepared using immunoprecipitation lysis buffer [20 mmol / L Tris - Cl (pH 7.5), 150 mmol / L NaCl, 10 % glycerol, and 1 % Triton X-100].

NHLF cells transfected with siRNA molecules were lysed in High Salt ELB lysis buffer [1 M Tris pH 8.0, 1 % NP - 40, 250 mM NaCl, 5 mM EDTA] supplemented with protease and phosphatase inhibitors (1x G - Biosciences Protease Arrest, 200 µM Na3VO4, and 1 mM PMSF).

Cell pellets were resuspended in 1 ml cell lysis buffer [5 mM Pipes pH 8.0, 85 mM KCl, 0.5 % NP - 40] containing protease inhibitors and incubated for 10 min on ice.

Cells were lysed with TN1 lysis buffer (50 mM Tris, 125 mM NaCl, 1 % Triton X-100, 10 mM EDTA, 10 mM sodium fluoride and 10 mM sodium pyrophosphate) supplemented with protease inhibitor cocktail (1.2 mM AEBSF, 0.46 μM aprotinin, 14 μM bestatin, 12.3 μM E-64, 112 μM leupeptin, 1.16 μM pepstatin)(Amresco; Solon, OH)-RRB- and 1 mM sodium orthovanadate (Enzo Life Sciences; Farmingdale, NY).

Make up «direct lysis buffer» by adding 1 mL of Proteinase K (e.g. Sigma P4850) to 100 mL DirectPCR Lysis Reagent (cell)(Viagen Biotech, Cat # 302 - C).

For this protocol, samples were lysed in 1 × RIPK2 lysis buffer [50 mM Tris (pH 7.5), 0 mM MgCl2, 1 % Triton X-100, 1 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA, with freshly added 1 mM β - glycerophosphate and protease inhibitors (phenylmethylsulfonyl fluoride and aprotinin)-RSB- and immunoprecipitated overnight using 1.5 μg rabbit anti-RIPK2 antibody.

At the end of the desired treatment times, cell lysates were prepared in lysis buffer (1 % NP - 40, 0.5 mM Tris - HCl (pH 7.5), 0.14 M NaCl, 5 mM KCl, 5 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride) plus serine / threonine phosphatase inhibitor cocktail.

After an overnight incubation at 4 ° C, for thorough stabilization, samples were homogenized in lysis buffer and total RNA was isolated using the GE Illustra RNAspin Isolation Kit (GE Healthcare) according to the manufacturer's protocol.

Hippocampus or frontal cortex samples were homogenized in lysis buffer (10 mm Tris, pH 7.5, 10 mm NaCl, 3 mm MgCl2, 1 mm EDTA, and 0.05 % NP - 40) with protease inhibitors (Halt protease inhibitor cocktail, Thermo Scientific) and protein concentration was determined by Bradford assay (Coomassie Plus, Thermo Scientific).

48 hours after transfection, cells were lysed with 100 µL / well 1 × passive lysis buffer (PLB, Promega) for 15 minutes with shaking.

Confluent 10 cm plates were fixed with 1 % PFA at room temperature for 10 minutes, lysed in SDS lysis buffer (50 mM Tris, 10 mM EDTA, 1 % SDS, Roche Complete protease inhibitors, pH 8.1), scraped and collected into 1.5 mL microcentrifuge tubes, and DNA was sonicated to 200 — 800 bp fragments with a Branson Sonifier 250 set to 30 % power / 90 % duty, four 10 second pulses.

Each single cell was carried through two rinses of PBS using a finely drawn glass capillary pipette under a dissecting microscope and then transferred in a minimal volume (0.5 µL) to a PCR tube containing lysis buffer, protease, tRNA and poly - T gripNA ™ probe (mTRAP ™ midi - kit, Active Motif, cat.

Tubes were spun, the nuclear lysis buffer with proteinase inhibitor was added and sonicated to form shared chromatin of about 200 — 1000 base pairs in length (data not shown).