All reagents are commercially sourced. Sources for basic reagents are provided in the supplemental information . Solarbio Technology Co., Ltd. provided the DEAE-Sepharose Fast Flow (Beijing, China). Sephacryl S200HR was purchased from Sigma-Aldrich Company (Darmstadt, Germany). CCK-8 Cell Counting Kit was purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). GenStar Biosolutions Co., Ltd. (Beijing, China) provided 2× Real Star Fast SYBR qPCR Mix kit. Applied Biological Materials Inc (British Columbia, VIC, Canada) provided All-In-One 5× RT MasterMix. Cell Signaling Technology (Boston, MA, USA) provided the rabbit mAbs for β-actin, phospho-NF-κB p65, HO-1, NQO1, and anti-rabbit IgG HRP-linked antibody. PRRS virus nucleocapsid protein mAb was purchased from GeneTex (SAN Antonio, TX, USA). Millipore (Waltham, MA, USA) provided the chemiluminescent HRT substrate. Both the DAPI staining solution and FITC-labeled goat anti-rabbit IgG (H+L) were bought from Beyotime Biotechnology Co., Ltd. in Shanghai, China.
Sephacryl s 200 hr
Manufactured by Merck Group
Sourced in Germany
Sephacryl S-200 HR is a size-exclusion chromatography medium used for the separation and purification of macromolecules, such as proteins, nucleic acids, and polysaccharides. It is a cross-linked copolymer of allyl dextran and N,N'-methylenebisacrylamide, which provides a porous matrix for the separation of molecules based on their size and molecular weight.
Automatically generated - may contain errors
Lab products found in correlation
Fitc labeled goat anti rabbit igg h l, by Beyotime (1 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
All in one 5 rt mastermix, by Applied Biological Materials (1 mentions)
Cck 8 cell counting kit, by Vazyme (1 mentions)
Dapi staining solution, by Beyotime (1 mentions)
Realstar fast sybr qpcr mix kit, by GenStar (1 mentions)
Amicon ultra, by Merck Group (1 mentions)
Alcohol dehydrogenase, by Merck Group (1 mentions)
Carbonic anhydrase, by Merck Group (1 mentions)
Beta amylase, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Iodoacetamide, by GE Healthcare (1 mentions)
Glycine, by GE Healthcare (1 mentions)
Dithiothreitol (dtt), by GE Healthcare (1 mentions)
Ipg strip, by GE Healthcare (1 mentions)
Methanol, by GE Healthcare (1 mentions)
Acrylamide, by GE Healthcare (1 mentions)
Bis acrylamide, by GE Healthcare (1 mentions)
Coomassie blue, by GE Healthcare (1 mentions)
Sodium hydrosulfite, by Merck Group (1 mentions)
8 protocols using sephacryl s 200 hr
1
PRRS Virus Nucleocapsid Protein Detection
2
Efficient EV Isolation and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The culture media was collected for isolation and purification of EVs using ultrafiltration coupled with size-exclusion chromatography (UF-SEC), as this combination of techniques ensures the efficient enrichment of small-sized EVs suitable for compositional and functional studies.42 (link),43 (link) First, the media was centrifuged for 10 min at 300xg to remove dead cells, for 10 min at 2500xg to remove cell debris and apoptotic bodies, and then filtered through a 0.2 μm membrane to remove large vesicles and aggregates. The media was concentrated using 100 kDa centrifugal ultrafiltration unit Amicon Ultra (UFC9100, Millipore/Sigma Aldrich) according to the instructions of the manufacturer. For EV enrichment, 1 ml of concentrated cell culture media from approximately six T150 flasks was subjected to SEC on a 30 cm length and 1.5 cm diameter Sephacryl S-200 HR (Sigma, GE17-0584-01) column at 4°C using sterile phosphate-buffered saline (PBS) as mobile phase. The absorbance of the collected fractions (approximately 30 fractions of 1 ml each) was measured at 280 nm to determine the EV-containing fractions, which were then further concentrated to a smaller volume, characterized, and used for subsequent studies.
3
Purification of Proteins by Size Exclusion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Size exclusion chromatography was done manually. A size exclusion column was packed using 45 ml of Sephacryl S-200 HR (Sigma-Aldrich) beads. The void volume (24 ml) of this column was determined by running blue dextran. 1mg of protein (in 100 μl volume) was loaded on the column equilibrated with the buffer containing 20mM HEPES-KOH pH7.6, 100mM KCl and 10% Glycerol. Fractions of 50 μl were collected and the absorbance at 280 nM were recorded. Protein fractions were also analyzed on 12% SDS-PAGE. Gel filtration standard consisted of a mixture of Beta-amylase (200 kDa), Alcohol Dehydrogenase (150 kDa), Bovine Serum Albumin (66 kDa) and Carbonic Anhydrase (29 kDa) bought from Sigma.
4
Protein Separation and Identification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sephacryl S-200 HR and sodium hydrosulfite were obtained from Sigma-Aldrich chemicals Co., Sweden. The disposable PD-10 columns, IPG strips, DTT, Iodoacetamide, Acrylamide, Bis-Acrylamide, Glycine, Methanol, Glacial Acetic acid, β-mercaptoenthanol and Coomassie blue, were obtained from GE Health Care. HNE was obtained from Cayman Chemical Company. Dialysis tubing was sourced from Spectrum laboratories, Inc. (Rancho Domingues, CA, USA). OFFGEL starter kit was procured from Agilent Technologies. All chemicals were of reagent grade or greater purity.
5
Characterizing SMA-PS Micelle Properties
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sephacryl-S200 HR (Sigma) gel chromatography of SMA-PS with or without human serum albumin (HSA) was carried out to determine apparent molecular size of SMA-PS micelles in an aqueous system, and examined the property of SMA for albumin binding. In brief, SMA-RB (2.0 mg/mL) was first dissolved in 0.2 M sodium phosphate buffer pH 7.4. Then, different amounts of HSA (0–10 mg/mL) were added and incubated for 30 min at room temperature. Then, 1.0 mL of each sample was subjected to a Sephacryl-S200 HR column (size: 1.5 cm Ø × 48 cm (h)), and eluted with 0.2 M sodium phosphate buffer (pH 7.4); a 3.2 mL fraction was collected from each tube, being monitored at 280 nm for HSA and 549 nm for RB micelles.
6
Lipid-based Nanoparticle Formulation Development
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dipalmitoylphosphatidyl choline (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N [amino (polyethylene glycol)-2000] (DSPE-PEG (2000)-NH2) were obtained from Avanti Polar Lipids Inc. (Alabaster, AL, USA, supplied by Labco LLC. Dubai, UAE). Cholesterol, calcein disodium salt, L-glutamine, antibiotic solutions (penicillin and streptomycin), trypsin, fetal bovine serum (FBS), RPMI-1640 medium, Dulbecco’s Phosphate Buffered Saline (DPBS) medium and the bicinchoninic acid (BCA) kit were obtained from Sigma Aldrich Chemie GmbH (Munich, Germany, supplied by Labco LLC. Dubai, UAE). Chloroform was obtained from Panreac Quimica S.A. (Spain). Doxorubicin-hydrochloride was obtained from Euroasian Transcontinental (Lower Parel, Mumbai, India). Sephadex G-100, Sephadex G-25, and Sephacryl S200 HR were obtained from Sigma-Aldrich (Munich, Germany, supplied by Labco LLC. Dubai, UAE). Trastuzumab (Herceptin) was obtained from Hoffmann-La Roche Limited (Basel, Switzerland, supplied by Aster pharmacy, Sharjah, UAE). 2,4,6 trichloro-1,3,5 triazine (cyanuric chloride) was obtained from Sigma-Aldrich (St. Louis, MO, US, supplied by LABCO LLC. Dubai, UAE). SKBR3 (HER2 + cells) and MDA-MB-231 (HER2- cells) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).
7
Purification of Myrosinase Enzyme
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chromatographic runs were performed in a BioLogic LP System equipped with a fraction collector and the LP Data View software (Bio-Rad, Hercules, CA, USA). A 0.7 × 30 cm column (Bio-Rad, Hercules, CA, USA) was used, which was packed with 15 mL of Sephacryl S-200 HR (Sigma Aldrich, Schnelldorf, Germany). The calibration curve was constructed using the protein gel filtration kit (13.5–75 kDa) (Sigma Aldrich, Schnelldorf, Germany). Chromatography was performed loading 1 mL of the previously purified myrosinase fraction (1 mg/mL), and it was eluted at a flow of 0.3 mL/min using 0.2 M Tris·HCl buffer, pH 7.4.
8
Recombinant Protein Purification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All chemicals used in this study were of analytical reagent grade. Ampicillin, isopropyl-β-D-1-thiogalactopyranoside (IPTG), phenyl sepharose, sephacryl S-200 HR, oligonucleotides and other chemicals were purchased from Sigma-Aldrich, Co. (St. Louis, USA). Restriction enzymes, DNA ladders, dNTPs mix and other material used for cloning were obtained from New England Biolabs Inc. (MA, USA). Ni-NTA agarose beads, SDS-PAGE molecular weight marker were purchased from GE Healthcare (UK). DNA gel extraction kit was procured from Qiagen Inc.(CA, USA).
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!