FLAG-TEV-SNAP-Ago2 expressed in HEK293T cells was labeled with SNAP-Surface Alexa Fluor 647 (NEB) and immunopurified by an anti-FLAG antibody conjugated onto Dynabeads Protein G (Invitrogen). The beads were washed three times by wash buffer, three times by wash buffer containing 2 mM ATP, and finally rinsed by lysis buffer. The beads were incubated with lysis buffer, crude lysates, or their boiled supernatants containing 2 U/μL TurboTEV protease (Accelagen) at room temperature for 1 hour. To prepare benzonase and proteinase K–treated (BZ/PK) supernatants, the supernatants were mixed with 1 U/mL benzonase (Millipore) and incubated at 37°C for 1 hour, then mixed with 2 mg/mL proteinase K and incubated at 37°C for 1 hour, and finally reboiled at 95°C for 15 minutes to deactivate proteinase K. The benzonase or proteinase K treatment was omitted for the PK or BZ condition, respectively.
Benzonase
Manufactured by Merck Group
Sourced in United States, Germany, Switzerland, United Kingdom, France, Denmark, Canada, Japan, Macao
Benzonase is a recombinant endonuclease enzyme that can degrade DNA and RNA. It is commonly used in various laboratory applications to remove nucleic acid contaminants from protein samples.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (59 mentions)
Protease inhibitor cocktail, by Merck Group (43 mentions)
Complete protease inhibitor cocktail, by Roche (31 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (28 mentions)
Protease inhibitor, by Roche (23 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (23 mentions)
Lysozyme, by Merck Group (20 mentions)
Pvdf membrane, by Merck Group (19 mentions)
Complete protease inhibitor, by Roche (19 mentions)
Ripa buffer, by Thermo Fisher Scientific (18 mentions)
Phosstop, by Roche (16 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (16 mentions)
Protein g dynabead, by Thermo Fisher Scientific (15 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (14 mentions)
Protease inhibitor, by Merck Group (13 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (12 mentions)
Ionomycin, by Merck Group (12 mentions)
L glutamine, by Thermo Fisher Scientific (12 mentions)
Dnase 1, by Thermo Fisher Scientific (11 mentions)
Anti flag m2 affinity gel, by Merck Group (11 mentions)
1 234 protocols using benzonase
1
Purification and Protease-Mediated Cleavage of FLAG-Tagged Proteins
2
Cytoplasm, Nucleus, and Chromatin Fractionation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cytoplasmic fraction was obtained by resuspending cells in buffer A (10 mM HEPES, 10 mM KCl, 340 mM sucrose, 10% glycerol, protease and phosphatase Inhibitors, and 2 mM EDTA). Triton X-100 was added to a final concentration of 0.1% and cells were incubated on ice for 5 min. Cells were then centrifuged at 350×g for 3 min. The remaining pellet (nuclei) was washed in buffer A without Triton X-100. Nuclei were ruptured by resuspending in buffer B (3 mM EDTA, 0.2 mM EGTA, 5 mM HEPES pH 7.9, protease and phosphatase inhibitors). The nuclear fraction was then centrifuged at 1700×g for 5 min and the supernatant was collected as the nuclear soluble fraction. The chromatin pellet was washed twice in Benzonase buffer (50 mM Tris–HCl pH 7.9, 50 mM NaCl, 5 mM KCl, 3 mM MgCl2, and protease and phosphatase inhibitors) and digested in Benzonase buffer, supplemented with 125 U of Benzonase (Merk Millipore) either at room temperature for 30 min or overnight rolling at 15 rpm at 4 °C. Samples were then centrifuged at 20,000×g for 5 min, the supernatant was collected as the chromatin soluble fraction.
3
Cell Lysis and Immunoprecipitation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested and lysed in NP-40 buffer (20 mM Tris·HCl pH 8.0, 137 mM NaCl, 1% NP-40, 10% glycerol, 2 mM EDTA, 1% protease inhibitor cocktail) for 30 min at 4 °C. After centrifugation, the supernatant was incubated with the indicated antibodies and protein G or A Sepharose slurry (GE Healthcare, USA) at 4 °C rotating overnight. For benzonase treatment, the supernatant was treated with benzonase (Millipore, USA) at 10 U/mL at 4 °C while rotating for 2 h before incubating with the antibodies. The beads were then washed and analyzed by immunoblotting.
4
Myc-Tagged Protein Immunoprecipitation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After washing with PBS, cells were resuspended in 500 μl of benzonase buffer (20 mM Tris-HCl at pH 7.5, 40 mM NaCl, 2 mM MgCl2, 0.5% NP-40, 50 U/ml benzonase [Millipore], 1× Protease inhibitor [Roche], 1× phosphatase inhibitor [Roche]), incubated for 10 min at 4 °C and cleared by centrifugation at 15,000 × g for 10min42 (link). The resultant extracts (WCE) were diluted with by adding 2 × WCE volume of No-salt IP buffer (25 mM Tris-HCl at pH 7.5, 1.5 mM DTT, 15% Glycerol, 1× Protease inhibitor, 1× phosphatase inhibitor). Twelve microliter of EZview Red anti-Myc affinity Gel (Sigma) were added to 300 µl of diluted extracts and incubated for 2 h at 4 °C. After incubation, beads were washed three times with IP buffer (25 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1.5 mM DTT, 10% Glycerol, 0.25% NP-40, 1× Protease inhibitor, 1× phosphatase inhibitor) and proteins were eluted with 50 μl of SDS sample buffer.
5
BRD4 Interactome Profiling by IP-MS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IP-MS experiments were performed as described by Arnold et al.47 (link). Briefly, the K562 dTAG-BRD4 cells were lysed in a native IP buffer (20 mM Tris pH 8.0, 50 mM NaCl, 0.5% (vol/vol) NP-40, 10% (vol/vol) glycerol, protease inhibitor cocktail (Roche), phosphatase inhibitor cocktail (Roche) supplemented with benzonase (Millipore, Cat.# E1014-25KU). Nuclei were isolated by centrifugation and lysed in native IP buffer supplemented with benzonase (Millipore, Cat.# E1014-25KU). For IP 10 µg of the BRD4-specific antibody (Bethyl, Cat.# A301-985A50) or of isotype-matched IgG (Bethyl, Cat.# P120-101) and 1,5 mg Dynabeads Protein G (Invitrogen, Cat.# 10004D) were used per sample. After two washes with the native IP buffer, three washes were performed with a washing buffer (20 mM Tris pH 8.0, 125 mM NaCl, 0.5% (vol/vol) NP-40). Five biological replicates were performed per condition. Mass spectrometric analysis on a Q-Exactive HF Orbitrap (Thermo Scientific) and processing of the data was done as described previously47 (link).
6
Benzonase Treatment for Particle Nucleic Acid Removal
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Equivalent protein amounts (0.8–3 × 1012 particles) of rsT1L or rsT3DIT1L1 virions or TC particles were diluted in benzonase buffer (50 mM Tris-HCl, 2 mM MgCl2, pH 8.0) and either mock-treated or treated with 1 U/µL of benzonase (Millipore, Burlington, MA, USA) at room temperature or at 37 °C for 1 h to remove extra-particle nucleic acids. Based on protein normalization, virions were diluted approximately 3- to 4-fold relative to TC particles. benzonase was inactivated with 0.5 M EDTA (pH 8.0), and RNA was extracted from virions and TC particles by TRIzol (Invitrogen, Waltham, MA, USA) extraction per manufacturer’s protocol. Concentration and quality of RNA were determined using a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and visualized as a gel display of electropherograms. Displays are automatically adjusted for fluorescence level so that RNA peaks were visible.
7
Benzonase® Assisted Depth Filtration
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Benzonase® (Sigma, St. Louis, MO, USA) was added to the bioreactor at 10 units/mL and incubated for 1 h before the clarification step. Depth filtration was evaluated as an alternative to the commonly used centrifugation as a cell removing step. From each harvest, 1 L of the harvested material after Benzonase® treatment was centrifuged at 4000× g for 10 min and another 1 L was filtered through MilliStack D0HC® (Millipore, Rockville, MD, USA) depth filter at 7.66 mL/min in sterile conditions as previously described [28 (link),29 (link)]. Before filtration, the depth filter was equilibrated with the filter buffer (50 mM HEPES, 300 mM NaCl at pH 7.2). The clarified material was supplemented with HEPES (Sigma, St. Louis, MO, USA) to a final concentration of 70 mM to maintain the pH at 7.2 during the inactivation. The material was inactivated by adding 0.1% (v/v) β-propiolactone (Sigma, St. Louis, MO, USA) and then stirred for 24 h at 4 °C. The inactivated supernatant was frozen at −80 °C and was thawed upon loading to the AKTATM Avant 25 system (Cytiva, Marlborough, MA, USA) for evaluation of the ion exchange chromatography membranes.
8
DNA2 and WRN Immunoprecipitation in HEK293T
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293T cells were transfected with empty vectors, FLAG-DNA2, and Strep-HA-WRN by calcium phosphate. 48 h after transfection, cells were treated with 4 mM HU for 3 h, lysed in benzonase lysis buffer (50 mM Tris-HCl, pH 7.5, 75 mM KCl, 2 mM MgCl2, 20 mM NaF, 10 mM β-glycerophosphate, 0.2 mM Na3VO4, and 0.2% Triton X-100) supplemented with protease inhibitors (EDTA-free tablet; Sigma-Aldrich) by passing 10 times through a 26-G syringe needle and incubated 1 h at 4°C with 2 U/µl benzonase (Sigma-Aldrich) to digest genomic DNA. KCl and EDTA concentrations were adjusted to 120 and 3 mM, respectively, and lysates were centrifuged at 14,000 rpm for 30 min. Immunoprecipitations of clarified lysates were performed with FLAG M2 or HA affinity agarose resin (Sigma-Aldrich) overnight at 4°C. Beads were washed 5 times with wash buffer (50 mM Tris-HCl, pH 7.5, 150 mM KCl, 3 mM EDTA, 2 mM MgCl2, 20 mM NaF, 10 mM β-glycerophosphate, 0.2 mM Na3VO4, and 0.2% Triton X-100) and bound proteins were eluted by boiling in SDS-PAGE sample buffer.
9
Thawing and Washing Frozen PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following media were prepared for thawing the frozen PBMCs: Medium A (RPMI, 30% Fetal Bovine Serum, 1:10,000 dilution Benzonase (Sigma), final concentration 2.5 units/mL) or Medium B (MarrowMax ™ (Thermo Fisher Scientific, Waltham, MA) with 1:10,000 dilution of Benzonase (Sigma), 2.5units/mL) and were warmed up to 37°C in a water bath.
Frozen PBMC samples (~1mL in 2mL cryotubes) were thawed in a 37°C water bath for 3 min and transferred into two separate 15mL Falcon tubes with ~ 500 ul volume of cell suspension. Thawing medium A or B was added drop-wise using a transfer pipette every 3-4 sec for the first 2 mL and then filled up to 10mL. Sample were washed a total of 3x by first centrifuging at 300xg for 10 min, removing supernatant, and re-suspending the pellet with 10mL of the corresponding thaw medium. 10 × 1mL cell aliquots were transferred into sterile Matrix ™ microtubes (Thermo Scientific ™ ) and incubated at 37°C/5% CO 2 for 1 hr. Cells were washed with prewarmed Medium C (RPMI 1640 with 15% FBS, 1% Pen/Strep). The 10 matrix tubes for each sample are pooled into a fresh, sterile, 15mL Falcon tube. Samples are washed 2x with medium as described above. Samples are realiquoted into10×1mL sterile matrix tubes.
Frozen PBMC samples (~1mL in 2mL cryotubes) were thawed in a 37°C water bath for 3 min and transferred into two separate 15mL Falcon tubes with ~ 500 ul volume of cell suspension. Thawing medium A or B was added drop-wise using a transfer pipette every 3-4 sec for the first 2 mL and then filled up to 10mL. Sample were washed a total of 3x by first centrifuging at 300xg for 10 min, removing supernatant, and re-suspending the pellet with 10mL of the corresponding thaw medium. 10 × 1mL cell aliquots were transferred into sterile Matrix ™ microtubes (Thermo Scientific ™ ) and incubated at 37°C/5% CO 2 for 1 hr. Cells were washed with prewarmed Medium C (RPMI 1640 with 15% FBS, 1% Pen/Strep). The 10 matrix tubes for each sample are pooled into a fresh, sterile, 15mL Falcon tube. Samples are washed 2x with medium as described above. Samples are realiquoted into10×1mL sterile matrix tubes.
10
Western Blot Analysis of PCN Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PCN were lysed in digestion solution with proteinase and phosphatases inhibitors, and benzonase solution (50 mM Tris pH = 8.0, 4 mM MgSO4 and 1x benzonase - Sigma-Aldrich). As before, protein concentration was measured using the Pierce™ BCA Protein Assay Kit.
10–25 µg of protein was loaded and run on 4–12% Bis-Tris gel (or 12% for LC3B) (Novex, ThermoFisher, Waltham, MA, USA) and transferred to nitrocellulose (or methanol-activated polyvinylidene fluoride for LC3B) membrane. Membranes were blocked in 5% milk Tris-buffered saline with tween (TBS-T), incubated overnight at 4 °C with first antibody, washed in TBS-T, incubated for 1 h at room temperature with secondary antibody in 5% milk and finally washed in TBS-T. Membranes were developed using Odyssey CLx (LI-COR, Cambridge, UK), and quantifications were performed using ImageJ or Image Studio Lite (LI-COR, Cambridge, UK), normalized to respective loading control (GAPDH, actin or tubulin) and, for starved conditions, normalized to respective fed condition.
10–25 µg of protein was loaded and run on 4–12% Bis-Tris gel (or 12% for LC3B) (Novex, ThermoFisher, Waltham, MA, USA) and transferred to nitrocellulose (or methanol-activated polyvinylidene fluoride for LC3B) membrane. Membranes were blocked in 5% milk Tris-buffered saline with tween (TBS-T), incubated overnight at 4 °C with first antibody, washed in TBS-T, incubated for 1 h at room temperature with secondary antibody in 5% milk and finally washed in TBS-T. Membranes were developed using Odyssey CLx (LI-COR, Cambridge, UK), and quantifications were performed using ImageJ or Image Studio Lite (LI-COR, Cambridge, UK), normalized to respective loading control (GAPDH, actin or tubulin) and, for starved conditions, normalized to respective fed condition.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!