After rinsing the columns with PBS, 2 ml of PFP were applied on top of a qEV column (Izon Science) and 0.5 ml fractions were collected. Four EV-rich fractions (7–10) were pooled and either analyzed directly (see below) or concentrated using an Amicon Ultra-4 10 kDa centrifugal filter device (Merck Millipore).
Amicon ultra 4 10 kda
Manufactured by Merck Group
The Amicon-Ultra 4 (10 kDa) is a centrifugal filter device used for the concentration and purification of macromolecules, such as proteins, peptides, and nucleic acids. It has a molecular weight cut-off of 10 kDa, allowing the retention of molecules larger than this size while the smaller molecules pass through the membrane.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Qev column, by Izon Science (1 mentions)
Superdex 200 10 30 gl, by GE Healthcare (1 mentions)
Vectastain abc standard kit, by Vector Laboratories (1 mentions)
Fusion solo 7s edge, by Vilber (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Western lightning plus ecl, by PerkinElmer (1 mentions)
5 protocols using amicon ultra 4 10 kda
1
EV Isolation and Characterization Protocol
2
Phage Characterization and Titer Quantification
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Two
types of viruses with different surface structures (the envelope-type
bacteriophage Φ6 and non-envelope-type bacteriophage Qβ)
were used as model viruses. A lysogeny broth (LB) (Formedium) medium
containing calcium chloride (2 mM) (Ca-added LB medium) was used to
prepare their stock suspensions. The respective bacteriophages were
infected after incubation at 37 and 30 °C for E. coli and P. syringae, respectively, until the logarithmic growth phase. For antiviral
activity analyses, the prepared stock suspensions were purified and
concentrated using an ultrafiltration device (Amicon Ultra-4,10 kDa,
Merck). The plaque assay, a common method for evaluating bacteriophages,
was used to analyze the viral infection titer.8 (link) The viral titer was estimated using the plaque-forming units (PFU).
Culture plates were prepared by adding 1.5% (wt vol–1) agar powder (FUJIFILM Wako Pure Chemical) into the Ca-added LB
medium; additionally, 0.6% (wt vol–1) agar powder
was added to the Ca-added LB medium as a top agar for the plaque assay.
The viral suspension purity (PFU mg-protein–1) is defined as the viral titer concentration of the
stock suspension divided by the protein concentration [(PFU mL–1)/(mg-protein mL–1)]. The protein concentrations of the stock suspensions were determined
by measuring their absorbance at 280 nm using a NanoDrop spectrophotometer
(ND-1000, Thermo Fisher Scientific).
types of viruses with different surface structures (the envelope-type
bacteriophage Φ6 and non-envelope-type bacteriophage Qβ)
were used as model viruses. A lysogeny broth (LB) (Formedium) medium
containing calcium chloride (2 mM) (Ca-added LB medium) was used to
prepare their stock suspensions. The respective bacteriophages were
infected after incubation at 37 and 30 °C for E. coli and P. syringae, respectively, until the logarithmic growth phase. For antiviral
activity analyses, the prepared stock suspensions were purified and
concentrated using an ultrafiltration device (Amicon Ultra-4,10 kDa,
Merck). The plaque assay, a common method for evaluating bacteriophages,
was used to analyze the viral infection titer.8 (link) The viral titer was estimated using the plaque-forming units (PFU).
Culture plates were prepared by adding 1.5% (wt vol–1) agar powder (FUJIFILM Wako Pure Chemical) into the Ca-added LB
medium; additionally, 0.6% (wt vol–1) agar powder
was added to the Ca-added LB medium as a top agar for the plaque assay.
The viral suspension purity (PFU mg-protein–1) is defined as the viral titer concentration of the
stock suspension divided by the protein concentration [(PFU mL–1)/(mg-protein mL–1)]. The protein concentrations of the stock suspensions were determined
by measuring their absorbance at 280 nm using a NanoDrop spectrophotometer
(ND-1000, Thermo Fisher Scientific).
3
Purification of p190-PH:RhoA•GTPγS Complex
4
Purification of recombinant human GBP1
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Full-length human GBP1 was cloned in pET-28a to generate an N-terminally His-tagged hGBP1 construct. pET-28a-hGBP1 was transformed into CleanColi BL21 (Lucigen), and the bacteria were grown in 2xYT medium until an OD600 of 0.5-0.7. Protein expression was then induced at 30 °C for 5 h with 0.2 mM IPTG. The bacterial pellet was resuspended in resuspension buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Tween 20) and frozen at −80 °C until purification. For most assay, protein was freshly purified on a Ni-NTA affinity column using standard protocols48 (link). Protein yield was quantified using Beer-Lambert law. After purification on a Ni-NTA columns, GBP1 was further purified on a size exclusion chromatography column (Superdex 200 10/30 GL, GE Healthcare) in running buffer (50 mM Tris pH 7.4, 150 mM NaCl) and concentrated using Amicon Ultra4 10 kDa (Millipore).
5
Western Blot Protein Detection Protocol
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The biotinylated proteins were separated by 5–20% SDS-PAGE, transferred to a nitrocellulose membrane, and detected using VECTASTAIN ABC Standard Kit (Vector Laboratories, Burlingame, CA, USA). For Western blotting, cells were lysed with TBS containing 0.5% nonidet P-40 and a protease inhibitor cocktail and sonicated. Proteins secreted into culture media were concentrated by Amicon Ultra-4 10 kDa (Millipore) after removing cell debris by centrifugation at 1000× g for 5 min. Protein concentrations in the cell lysates and media were measured using a Pierce BCA Protein Assay Kit (ThermoFisher Scientific), and the proteins were denatured in a Laemmli sample buffer. Proteins were separated by 5–20% SDS-PAGE, transferred to nitrocellulose membranes. After blocking with TBS containing 5% skim milk and 0.05% Tween-20, the membranes were incubated with 1st Ab and subsequently with HRP-labeled 2nd Ab. Signals were visualized with Western Lightning Plus-ECL (PerkinElmer, Waltham, MA, USA) or SuperSignal West Femto Maximum Sensitivity substrate (Thermo Fisher Scientific) using FUSION SOLO 7s EDGE (Vilber-Lourmat, Eberhardzell, Germany).
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