Surface proteins of CD were isolated using a “washing off” method employing 1 M LiCl [15 (link)]. Shortly, 1 g of bacterial mass was suspended in 7 mL of freshly prepared 1 M LiCl and incubated at room temperature (RT) for 1 h, with shaking. Then, the mixture was centrifuged at 6000× g for 5 min (Heraeus Contifuge Stratus, Thermo Fisher Scientific, Waltham, MA, USA). The supernatant was collected and dialyzed against water for at least 48 h with frequent water change. The protein solution was then concentrated with Amicon Ultra (Thermo Fisher Scientific, Waltham, MA, USA), MWCO = 10,000 Da. Protein concentration was estimated with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to instruction provided by supplier. The protein solution was aliquoted and stored at −20 °C until used.
Amicon ultra
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Amicon Ultra is a centrifugal ultrafiltration device used for the concentration and purification of macromolecules, such as proteins, enzymes, and antibodies. It features a regenerated cellulose membrane that allows for the selective retention of molecules based on their molecular weight.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Tcep disulfide reducing gel, by Thermo Fisher Scientific (2 mentions)
Heraeus contifuge stratus, by Thermo Fisher Scientific (1 mentions)
Helixate fs, by CSL Behring (1 mentions)
Sodium hypochlorite naocl, by Merck Group (1 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (1 mentions)
Protein a g agarose beads, by Thermo Fisher Scientific (1 mentions)
Accuspin 17r centrifuge, by Thermo Fisher Scientific (1 mentions)
7 protocols using amicon ultra
1
Isolation of Surface Proteins from CD
2
Sodium Hypochlorite Characterization Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sodium hypochlorite (NaOCl) was purchased from Sigma-Aldrich (St. Louis, MO). NaOCl concentration was determined using an extinction coefficient of 350 M−1 cm−1 at 292 nm in water. Zeba Spin desalting columns and Amicon Ultra centrifugal filters were purchased from Thermo Fisher Scientific (Waltham, MA) and Millipore Sigma (Burlington, MA), respectively. Helixate FS (CSL Behring, Kankakee, IL, USA; Lots 270PP4J and 27N1VK1) was purchased from the manufacturer and reconstituted using sterile water in accordance with the kits and instructions provided.
3
Anti-HPi2 Antibody Purification Protocol
4
Ubiquitin–Peptide Conjugation via Bpy Cross-Linking
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The purified protein was subjected to buffer-exchange
into 50 mM ammonium bicarbonate containing 5 mM EDTA, pH 8.5, using
a 3,000 MW cutoff Amicon Ultra (Fisher Scientific) centrifugal filter
unit and concentrated to 1 mg/mL. The solution was treated with an
equal volume of immobilized TCEP disulfide reducing gel (Thermo Scientific)
for 2 h before incubating with 5 equiv of Bpy in 30% acetonitrile/50
mM ammonium bicarbonate buffer, pH 8.5, for 2 h. Afterward, excess
reagents were removed through buffer exchange to yield the Bpy-cross-linked
ubiquitin–peptide conjugates, which were then used directly
in subsequent studies without further purification.
into 50 mM ammonium bicarbonate containing 5 mM EDTA, pH 8.5, using
a 3,000 MW cutoff Amicon Ultra (Fisher Scientific) centrifugal filter
unit and concentrated to 1 mg/mL. The solution was treated with an
equal volume of immobilized TCEP disulfide reducing gel (Thermo Scientific)
for 2 h before incubating with 5 equiv of Bpy in 30% acetonitrile/50
mM ammonium bicarbonate buffer, pH 8.5, for 2 h. Afterward, excess
reagents were removed through buffer exchange to yield the Bpy-cross-linked
ubiquitin–peptide conjugates, which were then used directly
in subsequent studies without further purification.
5
Extraction and Quantification of IgA/IgG
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total IgA or IgG was extracted from 25 – 100mL of milk or 1mL of plasma using peptide M or protein A/G agarose beads (Thermo), respectively, following manufacturer’s protocols. Ig was concentrated using Amicon Ultra centrifugal filters (10 kDa cutoff; Fisher) following manufacturer’s protocol, and quantified by Nanodrop.
6
Determining Drug Encapsulation in Solid Lipid Nanoparticles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The drug content and loading in the TA-SLNs was determined by using the lipid precipitation method where, at first, the lipid in the SLN and SLN-IG (0.1 mL) was precipitated using 0.9 mL of methanol, followed by centrifugation (AccuSpin 17R centrifuge, Fisher Scientific, Hanover, IL, USA) under 13,000 rpm for 15 min and analyzing the supernatant using HPLC.
Drug loading in SLNs were calculated using following formula: where Wt is the total weight of the drug, Wu is the weight of the unentrapped drug, and WL is the weight of the lipids.
The EE(%) in TA-SLNs were calculated by estimating the concentration of the free drug in the aqueous phase of an undiluted formulation, using an ultrafiltration method with 100-kDa centrifugal filter unit (Amicon Ultra, Hanover, IL, Fisher Scientific, USA). Briefly, a 500 uL aliquot of the formulation was added to the filter unit and centrifuged under 13,000 rpm for 15 to 20 min. The filtrate was further diluted and analyzed for the drug content using HPLC-UV system. The EE was calculated by using the Equation (2): where Di is the total drug content and Df is the free drug present in the aqueous phase.
Drug loading in SLNs were calculated using following formula: where Wt is the total weight of the drug, Wu is the weight of the unentrapped drug, and WL is the weight of the lipids.
The EE(%) in TA-SLNs were calculated by estimating the concentration of the free drug in the aqueous phase of an undiluted formulation, using an ultrafiltration method with 100-kDa centrifugal filter unit (Amicon Ultra, Hanover, IL, Fisher Scientific, USA). Briefly, a 500 uL aliquot of the formulation was added to the filter unit and centrifuged under 13,000 rpm for 15 to 20 min. The filtrate was further diluted and analyzed for the drug content using HPLC-UV system. The EE was calculated by using the Equation (2): where Di is the total drug content and Df is the free drug present in the aqueous phase.
7
Ubiquitin-Peptide Conjugation Workflow
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!