The eluted fractions containing the rYGP40 analysis protein were pooled, and the obtained protein solution was diluted 10 times with renaturation buffer (100 Mm phosphate buffer, 100 mM NaCl, 10% v/v glycerol, 2% v/v Brij® 35, Sigma Aldrich) (50 mM Tris-HCl of pH 8.5; 100 mM NaCl, 0.2% polyethylene glycol lauryl ether (Brij® 35, Sigma Aldrich), 10% glycerol) to eliminate the influence of 8 M urea. The renaturation process was carried out overnight at 4 °C with gentle mixing. The protein solution was concentrated 10 times on Amicon® Ultra-4 10 K Centrifugal Filter Devices (Merck Millipore), dialyzed against water, and lyophilized.
Amicon ultra 4 10 k centrifugal filter devices
Manufactured by Merck Group
Sourced in Germany
The Amicon® Ultra-4 10 K Centrifugal Filter Devices are laboratory equipment designed for sample concentration and buffer exchange. These devices utilize a centrifugal force to separate and concentrate macromolecules, such as proteins, from solutions through a selective membrane with a molecular weight cutoff of 10,000 Daltons.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Brij 35, by Merck Group (1 mentions)
Talon metal affinity resin, by Takara Bio (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
B per bacterial protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Hispur ni nta, by Thermo Fisher Scientific (1 mentions)
Mascot sequence matching software, by Matrix Science (1 mentions)
3 protocols using amicon ultra 4 10 k centrifugal filter devices
1
Renaturation and Purification of rYGP40
2
Purification and Labeling of EGFP-NT-Lysenin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EGFP-tagged nontoxic-type earthworm toxin lysenin (EGFP-NT-lysenin) was prepared as described previously (20 (link)) with some modifications. Briefly, Escherichia coli BL21(DE3) pLysS strain transformed with pET28-His6-EGFP-NT-Lys was induced to express EGFP-NT-lysenin by adding 1 mM isopropyl-β-d -thiogalactopyranoside for 4 h at 37°C. The cells were lysed with an X-tractor buffer kit (TaKaRa Bio). The lysate was applied to a column filled with TALON metal affinity resin (TaKaRa Bio). After washing the resin, EGFP-NT-lysenin was eluted with elution buffer (50 mM sodium phosphate, 300 mM sodium chloride, and 150 mM imidazole) and was concentrated in PBS by ultrafiltration using Amicon Ultra-4 10K centrifugal filter devices (Merck Millipore, Burlington, MA). NT1, SMS1KO22, SMS1KO22/SMS1-WT, SMS1KO22/SMS1-H328A, or SMS1KO22/SMS2 cells were detached using trypsin-EDTA (0.05%; Thermo Fisher Scientific) and reacted with 20 μg/mL EGFP-NT-lysenin for 30 min at 4°C. Untreated cells were used as controls. After washing with PBS, the cells were analyzed using a BD FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ).
3
Recombinant Schistosome Antigens Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The recombinant schistosome antigens were each transformed into competent E. coli BL21(DE3), as previously described, then grown in Overnight Express™ Instant terrific broth medium (Novagen, Germany) for 36 h at 26 °C at 300 rpm. Cultures were harvested by centrifugation and treated with 500 µL B-PER™ bacterial protein extraction reagent (Thermo Fisher Scientific, USA) and 1.0 μL Lysonase™ bioprocessing reagent (Novagen, Germany). The recombinant antigens were purified by IMAC using HisPur TM Ni-NTA (Thermo Scientific, USA), followed by ultrafiltration using Amicon® Ultra-4 10K centrifugal filter devices (Merck Millipore, Germany) with 20 mM potassium phosphate (KH 2 PO 4 -K 2 HPO 4 ) buffer (pH 7.5). The presence of purified antigens was confirmed by SDS-PAGE (sodium dodecyl sulfate -polyacrylamide gel electrophoresis) stained with Coomassie blue. The purified recombinant proteins were sent to Apical Scientific Sdn., Bhd. Mass for sequencing by MALDI-ToF MS/MS and spectral data were analyzed using Mascot sequence matching software (Matrix Science, USA) with MSPnr100 database and MS-Bridge protein prospector (http://prospector.ucsf.edu/ prospector/cgi-bin/msform.cgi?form=msbridgestandard).
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!