Two approaches were tested. A) Centricon using Amicon® Ultra Centrifugal Filters (Merck Millipore Ltd, MA), with a 3 kDa MWt cut-off: 100 µL of PBS or plasma containing CFH alone, Hb alone or a mixture of both were added to Centricons and centrifuged at 14,000×g, 4 °C, 2 h. B) Desalting via Micro Bio-Spin™ size-exclusion columns (Bio-Rad, CA), 6 kDa exclusion limit. 100 µL of PBS or plasma with oxyHb and CFH were added to columns and centrifuged at 1500×g, 22 °C for 2 min. Total heme level was determined on pre- and post- Centricon or pre- and post- size-exclusion columns to calculate the CFH by QuantiChrom™ Heme assay kit.
Micro bio spin size exclusion columns
Manufactured by Bio-Rad
Micro Bio-Spin size exclusion columns are a laboratory tool used for the separation and purification of biomolecules. They work on the principle of size-exclusion chromatography, allowing smaller molecules to pass through the porous matrix while larger molecules are retained. These columns are designed for quick and efficient sample preparation prior to downstream analysis or further processing.
Automatically generated - may contain errors
Lab products found in correlation
Wizard 3 1480 automatic gamma counter, by PerkinElmer (2 mentions)
Amicon ultra centrifugal filter, by Merck Group (1 mentions)
Carbonic anhydrase, by Merck Group (1 mentions)
Superdex 200 10 30 gl column, by GE Healthcare (1 mentions)
Kta purifier, by GE Healthcare (1 mentions)
Beta amylase, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Itlc strips, by Mirion Technologies (1 mentions)
Bio gel p 6, by Bio-Rad (1 mentions)
Herceptin, by Genentech (1 mentions)
Synergy h1 hybrid microplate reader, by Agilent Technologies (1 mentions)
96 well plate, by Greiner (1 mentions)
7 protocols using micro bio spin size exclusion columns
1
Purifying Hemoglobin and Complement Factor H
2
Site-specific Labeling of A11 cMb with Maleimide-Cy5.5
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A11 cMb was site-specifically labeled with maleimide-Cy5.5 (mal-Cy5.5) by selective reduction of and conjugation to C-terminal cysteines. In a typical reaction, 200 µg of protein at 1 mg/mL in phosphate-buffered saline (PBS) was reduced using a 2-fold molar excess of tris(2-carboxyethyl)phosphine (TCEP, Pierce) for 30 min at room temperature. Equimolar mal-Cy5.5 (Amersham, GE Healthcare) was added to the reduced A11 cMb for 2 h at room temperature. Excess mal-Cy5.5 was removed using a Micro Bio-Spin™ Size Exclusion Columns (Bio-Rad) pre-equilibrated with PBS. Dye-to-protein ratio (D:P) was determined by measuring the protein (280 nm) and Cy5.5 absorbance (675 nm) with a spectrophotometer (NanoDrop 2000). Successful conjugation was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC).
For SEC, a Superdex 200 10/30 GL column (GE Healthcare) was used with an ÄKTA purifier (GE Healthcare) with PBS as the mobile phase (0.5 mL/min). Absorbance at 280 nm (protein) and 675 nm (Cy5.5) was recorded. The following protein standards were used: beta-amylase (200 kDa), bovine serum albumin (66 kDa), and carbonic anhydrase (29 kDa) (Sigma).
For SEC, a Superdex 200 10/30 GL column (GE Healthcare) was used with an ÄKTA purifier (GE Healthcare) with PBS as the mobile phase (0.5 mL/min). Absorbance at 280 nm (protein) and 675 nm (Cy5.5) was recorded. The following protein standards were used: beta-amylase (200 kDa), bovine serum albumin (66 kDa), and carbonic anhydrase (29 kDa) (Sigma).
3
Radiolabeling of Obinutuzumab-based Cys-diabody
4
Radiolabeling and Characterization of A2cDb
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
[18F]SFB was resuspended in sodium borate buffer (33 MBq) in 25 μL SBB, 50 μM, pH 8.7 and incubated with A2cDb (50 μg in 50 μL SBB) for 10 min at 34 °C. Conjugated [18F]FB-A2cDb was purified using a Micro Bio-Spin size exclusion columns (BioRad) that was pre-blocked with PBS, 1% fetal bovine serum (FBS) as previously described.33 (link) Labelling efficiency and radiochemical purity were determined using ITLC strips (Biodex Medical Systems) with saline as solvent and analyzed by gamma counting (Wizard 3′ 1480 Automatic Gamma Counter, PerkinElmer). The immunoreactive fraction of radiolabeled A2cDb was measured by incubation with excess PSCA-positive cells (22Rv1-PSCA) and control cells (22Rv1) for 1 h at room temperature. Supernatant and cell-bound fractions were analyzed by gamma counting as previously described.34 (link)
5
Laccase-Catalyzed Tyrosine Nitration and ABTS Adduct Formation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A volume of 100 μL 10 μM laccase in 500 mM sodium citrate pH 5, containing 150 mM sodium nitrite and 150 mM hydrogen peroxide (both added in three successive steps at two hours’ interval) which produce peroxynitrite, was incubated 10 hours in order to nitrate the tyrosine residues of the protein. After salts removal by micro Bio-Spin size exclusion columns (Bio-Rad), the laccase assessed for its activity and was treated with ABTS in order to test its ability to form adducts.
The ABTS-tyrosine adduct was obtained by mixing 500 μM tyrosine and 500 μM ABTS+● in presence of catalytic laccase (7 nM) was incubated overnight in 50 mM ammonium acetate pH 5 and then analyzed by HPLC-MS system using the same method as previously described. A solution of 500 μM (tyrosine concentration) stoichiometric mixture of the aminoacids which from the identified modified peptide (TANNTNPY TNPPNTGVIR) with and without tyrosine was incubated with 500 μM ABTS+● and the reaction was monitored overnight in 20 mM sodium citrate pH 4.5 and in the presence of a catalytic amount of laccase (7 nM). Bovine serum albumin (BSA) was also used as control test, in the same manner.
The ABTS-tyrosine adduct was obtained by mixing 500 μM tyrosine and 500 μM ABTS+● in presence of catalytic laccase (7 nM) was incubated overnight in 50 mM ammonium acetate pH 5 and then analyzed by HPLC-MS system using the same method as previously described. A solution of 500 μM (tyrosine concentration) stoichiometric mixture of the aminoacids which from the identified modified peptide (TANNTNP
6
Trastuzumab Purification and Preparation
7
HOCl Exposure Effects on YbbN Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purified YbbN, at a final concentration of 20 mM in TE buffer (50 mM Tris-HCl [pH 8.0], 2 mM EDTA), was treated with 2 mM HOCl at room temperature for various lengths of time. HOCl was then removed with Micro Bio-Spin size-exclusion columns (BioRad) in accordance with the manufacturer's protocol. Purified YbbN (final concentration of 20 mM) was diluted in TE buffer containing various concentrations of HOCl. After 10 min at room temperature, HOCl was removed with Micro Bio-Spin size-exclusion columns.
Growth curves and dilutions on plates Cells were grown in 5 mL M9 + glucose at 37 C until OD 600 nm = 0.3. For growth curves, cells were treated with 2 mM HOCl and transferred to a 96-well plate (Greiner Bio-One). Growth was monitored for 8 h (OD 600 nm ) in a Biotek Synergy H1 Hybrid microplate reader. For dilutions on plates, cells were grown in 5 mL M9 + glucose until OD 600 nm = 0.3 and treated with 2 mM HOCl for 15 min. Serial dilutions were prepared in fresh M9 + glucose and 5 mL drops were pipetted onto LB agar plates. Plates were incubated overnight at 37 C.
Growth curves and dilutions on plates Cells were grown in 5 mL M9 + glucose at 37 C until OD 600 nm = 0.3. For growth curves, cells were treated with 2 mM HOCl and transferred to a 96-well plate (Greiner Bio-One). Growth was monitored for 8 h (OD 600 nm ) in a Biotek Synergy H1 Hybrid microplate reader. For dilutions on plates, cells were grown in 5 mL M9 + glucose until OD 600 nm = 0.3 and treated with 2 mM HOCl for 15 min. Serial dilutions were prepared in fresh M9 + glucose and 5 mL drops were pipetted onto LB agar plates. Plates were incubated overnight at 37 C.
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!