The chromophore AE of LDM was separated through a C4 column (150 × 10 mm; Phenomenex, Torrance, CA, USA) by HPLC. Then, the AE‐containing solution was mixed with the anti‐CD30‐LDP solution at a 1 : 3 molecular ratio and incubated at 4 °C for 12 h by gently shaking to form the enediyne‐integrated ADC anti‐CD30‐LDM. Next, free AE was removed by ultrafiltration centrifugation. The composition of the ADC was finally confirmed by reverse‐phase HPLC using a C4 column (250 × 4.6 mm; Phenomenex).
C4 column
Manufactured by Phenomenex
Sourced in Italy, United States
The C4 column is a type of high-performance liquid chromatography (HPLC) column used for the separation and purification of various chemical compounds. It features a stationary phase with a C4 (butyl) functional group, which allows for the separation of moderately hydrophobic molecules. The C4 column is commonly used in a wide range of applications, including pharmaceutical, biochemical, and environmental analysis.
Automatically generated - may contain errors
Lab products found in correlation
Hplc system, by Agilent Technologies (2 mentions)
Waters 2487 detector, by Waters Corporation (1 mentions)
Enterokinase, by New England Biolabs (1 mentions)
Snakeskin dialysis tubing, by Thermo Fisher Scientific (1 mentions)
Openlab cds software, by Agilent Technologies (1 mentions)
Prostar system, by Agilent Technologies (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Ps3 peptide synthesizer, by Protein Technologies (1 mentions)
C8 column, by Phenomenex (1 mentions)
2690 hplc, by Waters Corporation (1 mentions)
Mini protean 2, by Bio-Rad (1 mentions)
Precision plus protein kit, by Bio-Rad (1 mentions)
Pro q emerald 300 glycoprot probes kombo, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Breeze instrument, by Waters Corporation (1 mentions)
α cyano 4 hydroxycinnamic acid, by Shimadzu (1 mentions)
Axima performance, by Shimadzu (1 mentions)
15 protocols using c4 column
1
Enediyne-integrated ADC Preparation
2
Optimizing Disulfide-Bond Formation in Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
hCox17 at 1 mg/mL was reduced in 50 mM Tris pH 8.4, NaCl 100 mM and 200 mM DTT over night at room temperature. To initiate folding, samples were loaded in a PD-10 desalting column previously equilibrated with 50 mM Tris pH 8.4, NaCl 100 mM. Selected concentrations of GSH, Glycerol, TFE and Mia40 were added to the collected fraction to modulate or enhance the folding kinetics. To follow the oxidative reaction, removed aliquots at selected times were quenched with 2% TFA and loaded in a Waters 2690 HPLC system with a C4 column (Phenomenex) 250 × 4.6 5
mm. A linear gradient of 20–35% of 0.1% TFA in acetonitrile was applied for 60 minutes at a flow-rate of 0.75 ml/min, Eluted species were monitored by a coupled Waters 2487 detector at 214 nm and 280 nm wavelength. In the same way, a preparative VYDAC protein C4 was used to purify the intermediate species of the reaction. An increased 3 mL/min flow was applied to the same linear gradient of acetonitrile as above for 120 minutes. The purified species were identified by mass spectrometry and the reaction intermediate were quantified for the stop/go experiments in 50 mM Tris pH 8.4 NaCl 100 mM at 0.5 mg/mL14 (link),22 (link).
mm. A linear gradient of 20–35% of 0.1% TFA in acetonitrile was applied for 60 minutes at a flow-rate of 0.75 ml/min, Eluted species were monitored by a coupled Waters 2487 detector at 214 nm and 280 nm wavelength. In the same way, a preparative VYDAC protein C4 was used to purify the intermediate species of the reaction. An increased 3 mL/min flow was applied to the same linear gradient of acetonitrile as above for 120 minutes. The purified species were identified by mass spectrometry and the reaction intermediate were quantified for the stop/go experiments in 50 mM Tris pH 8.4 NaCl 100 mM at 0.5 mg/mL14 (link),22 (link).
3
Recombinant Ameloblastin Protein Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant mouse ameloblastin (rAmbn) and the variant delta 5 (AmbnΔ5), which lacks the sequence encoded by Exon 5, were expressed and purified following the method described in our recent publication (Su et al. 2019b (link)). Briefly, Ambn was expressed in Escherichia coli with Histidine (His), Thioredoxin (Trx), and S-tags in BL21, using pET-32a plasmid (Novagen) with inserted mouse Ambn gene (GenBank No. AAB93765.1). The expressed protein was purified using Nickel affinity chromatography (Ni-NTA Agarose, Qiagen), followed by dialysis through 10K MWCO dialysis membrane (SnakeSkin™ Dialysis Tubing, ThermoFisher). His-, Trx-, and S-tags were cleaved by Enterokinase (light chain, New England Biolabs), and were removed by a reversed phase High Performance Liquid Chromatography (HPLC) system (Varian Prostar system with Agilent OpenLab CDS software). Phenomenex C4 column (10 × 250 mm, 5 μm) was used, and proteins were eluted with an increasing gradient of 32–72% acetonitrile over 80 min, at a flow rate of 1.5 ml/min. Collected proteins were lyophilized and characterized with SDS-PAGE. The heat denaturation of rAmbn was performed by heating rAmbn at 95°C for 10 min.
4
HPLC Separation of Biological Samples
5
Recombinant Porcine Amelogenin Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Wild type (rP172) and variant recombinant porcine amelogenin
proteins were expressed in E. coli strain BL21(DE3)pLysS
(Stratagene, CA), and precipitated by 20% ammonium sulfate following the method
described previously [19] , [20] . The ammonium sulfate precipitate was dissolved in
water containing 0.1% TFA and purified using a high performance liquid
chromatography (HPLC) system (Varian, CA) equipped with a Phenomenex C4 column
(10×250 mm, 5 µm). The purified proteins
were lyophilized, kept at −20 °C, and dissolved in water before
use. Our group designed three double-variant strains of amelogenin; rP172(W45Y,
W161Y) will be referred to as rP172(W25), rP172(W25Y, W161Y) will be referred to
as rP172(W45), and rP172(W25Y, W45Y) will be referred to as
rP172(W161) [21] (link). The
positions of tryptophan residues of rP172 and its variants are illustrated in
Table
1 .Table 1.
proteins were expressed in E. coli strain BL21(DE3)pLysS
(Stratagene, CA), and precipitated by 20% ammonium sulfate following the method
described previously [19] , [20] . The ammonium sulfate precipitate was dissolved in
water containing 0.1% TFA and purified using a high performance liquid
chromatography (HPLC) system (Varian, CA) equipped with a Phenomenex C4 column
(10×250 mm, 5 µm). The purified proteins
were lyophilized, kept at −20 °C, and dissolved in water before
use. Our group designed three double-variant strains of amelogenin; rP172(W45Y,
W161Y) will be referred to as rP172(W25), rP172(W25Y, W161Y) will be referred to
as rP172(W45), and rP172(W25Y, W45Y) will be referred to as
rP172(W161) [21] (link). The
positions of tryptophan residues of rP172 and its variants are illustrated in
1
The tryptophan residues of wild type amelogenin and its
variants.
| Name | Location of Tryptophan residues | ||
|---|---|---|---|
| rP172 | W25 | W45 | W161 |
| rP172(W25) | W25 | ||
| rP172(W45) | W45 | ||
| rp172(W161) | W161 | ||
6
Recombinant Amelogenin Protein Expression
7
Recombinant FAS1 Domain Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant 4th_FAS1 domains of the WT, H572R and R555W TGFβIp proteins were cloned, expressed and purified as described previously21 (link). Briefly, the TGFBI genes were cloned in pCDF2 vectors and expressed in E.coli BL21DE3 cells. The proteins were purified by affinity chromatography using a manually prepared column with High Performance Ni-sepharose beads. Further purification was done using RP-HPLC (C4 column, Phenomenex) with a linear gradient of 50% acetonitrile in 0.01% trifluoroacetic acid at a flow rate of 3 ml/min. The purified proteins were lyophilized and stored in −20 °C.
8
Synthesis and Characterization of Peptide-Lipid Conjugates
9
Protein Desalting for Mass Spectrometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Desalted protein for mass spectrometry identification was obtained using a linear gradient of 0.1% TFA (solvent A) and acetonitrile containing 0.1% TFA (solvent B) from 5 to 65% of solvent B over 60 min, at a flow rate of 1.0 mL/min, on a C-4 column (250 mm × 4.6 mm, 5 μm particle size; 300 Å; Phenomenex, Castel Maggiore, Bologna, Italy). Elution was monitored at 214 nm.
10
Peptide Separation by Reversed-Phase HPLC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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!