Sentences with phrase «lysis buffer»

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

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.

Lyse cells with as small a volume volume of RIPA lysis buffer as possible before diluting the lysates with 1x PBS to the desired final volume.

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.

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].

Using a multichannel pipette, transfer 25 µL / well of direct lysis buffer (from Step 2) to the required number of 96 well PCR plates.

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.

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).

Nuclei were pelleted by centrifugation (5000 rpm for 5 min) and resuspended in 350 µl nuclear lysis buffer [50 mM Tris pH 8.1, 10 mM EDTA, 1 % SDS] containing protease inhibitors.

Cells were then lysed by adding 50 µL Lysis Buffer (provided).

The nuclear pellet was resuspended in a second lysis buffer containing 50 mM HEPES (pH 7.9), 0.4 M sodium chloride, and 1 mM EDTA, with protease inhibitors (20 µg / ml aprotinin, 5 µg / ml leupeptin, and 1 mM PMSF).

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).

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).

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.

Red blood cells were lysed in ACK lysis buffer (0.83 % NH4Cl, 0.5 % KHCO3, 0.5 μM EDTA) for 2 min and then neutralized with PBS or media.

Using a multichannel pipette, transfer 5µL of cells from each well of the 96 well cell culture plate into a PCR plate containing 25 µL / well of «direct lysis buffer» (from step 3).

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

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).

Single cells were washed once in PBS immediately following sorting and picked into PCR tubes containing lysis buffer with tRNA and flash frozen on dry ice prior to storing at − 80 °C.

Gastrocnemius muscles from IL - 15Rα — KO and B6129 mice were homogenized using a mechanical homogenizer (Polytron) in lysis buffer (50 mM Tris - HCl pH 8.0, 150 mM NaCl, 1 % IGEPAL 630, 2 mM EDTA, protease inhibitor).

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.

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).

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).

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.

The samples were diluted to 1 mg protein / ml with lysis buffer, and then diluted 1:1 with ELISA sample buffer.

iPS - RPE cells were collected on ice in lysis buffer (10 mM HEPES, 1 % Triton, 150 mM KCl, 1 mM PMSF, 10 ng / ml leupeptin, 1 mM DTT, 50 ng / ml aprotinin, 10 mM NaF, and 100 µM sodium vanadate) and incubated for 30 min on a tube rotator at 4 °C.

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.

The biotinylated anti-Argonaute antibody (12.5 μg; biotin was conjugated to an anti-human Ago antibody (# 01 - 2203, Wako, Japan)-RRB-(Hendrickson et al., 2009) was mixed with 250 μl Dynal M - 280 Streptavidin coated magnetic beads (Invitrogen), which were equilibrated by washing three times with lysis buffer and twice with PBS.

Single embryos were picked and crushed with a 26 - G needle into 200 μl of lysis buffer (supplemented with Proteinase K) from the Agencourt RNAdvance Tissue kit (Beckman Coulter, Cat.