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Antibodies and Antisera
Published in Lars-Inge Larsson, Immunocytochemistry: Theory and Practice, 2020
Alternatives to affinity purification include the labeled antigen methods (see Chapter 3, Section III) and, of course, preparation of monoclonal antibodies. It is practical to test polyclonal antisera on tissue as whole antisera first. Thus, in most applications, chances are great that totally unpurified antisera can be used (if supported by adequate controls). It is indeed a major mistake to begin a long and laborious purification scheme without first testing the serum directly. Methods for purifying IgG out of immune sera should be employed only if it is necessary to avoid other serum components or enzymes. Usually, this is not the case and crude serum may serve equally well or better than overtly manipulated IgG preparations. In certain applications, however, serum enzymes may destroy tissue-bound antigens, and, in other applications, the primary antibodies may have to be directly labeled or transformed into F(ab)2 or Fab fragments. In all of these cases, purification of IgG fractions is necessary. A method based on protein A-Sepharose,® as developed by Goding, is given in the Appendix.23
Combined Purging Approaches in Autologous Transplantation
Published in Adrian P. Gee, BONE MARROW PROCESSING and PURGING, 2020
Elizabeth J. Shpall, Robert C. Bast, Charles S. Johnston, William P. Peters, Roy B. Jones
The purification of the antibodies was carried out using five single-use protein A columns. Twelve milliliters of Protein-A Sepharose (Pharmacia LKB, Uppsala, Sweden) was packed in a 1.5 × 20 cm column (Bio-Rad Laboratories, Richmond, CA). The column was washed three times with 25 ml of sterile sodium citrate buffer (pH 3.0) and then neutralized by washing three times with 25 ml of sterile sodium phosphate buffer (pH 8.0).
Identification and Enrichment of Proteolytic Enzymes of IL-2 Activated Rat Natural Killer (A-NK) Cells: Potential Physiological Roles in NK Cell Function
Published in Ronald H. Goldfarb, Theresa L. Whiteside, Tumor Immunology and Cancer Therapy, 2020
Richard P. Kitson, Ken Wasserman, Ronald H. Goldfarb
The aggregate Boc-FSR AMCase and Suc-AAF AMCase activity from sucrose gradients were further purified over Sephacryl S-300 and heparin-Sepharose columns with a final purification factor of 6000-8000 fold. We believe that we have purified this enzyme to apparent homogeneity since even though three bands are visible on a modified SDS PAGE gel, all three bands are catalytically active. The molecular weight of the complex was determined to be approximately 7000,000 by molecular sieving on a calibrated column of Sephaccryl S-300. The biochemical properties of A-NKP-1 and A-NKP-2, including sensitivities to salts and detergents, coupled with their high molecular weight and substrate specificities led us to consider that this protease complex was related to MCP. The possibility that our enzymes were possibly related to MCP was intriguing since this complex appears to be ubiquitous in all cells examined to date, but which had heretofore been ignored in studies of lymphocyte proteases. We were therefore excited by the possibility that a “ubiquitous” enzyme system might play a specialized role in a specialized cell type: A-NK cells. In collaboration with Dr. A.J. Rivett (U. of Leicester), we have determined that our highly purified rat A-NK protease complex is cross reactive with anti-rat liver (MCP on a Western blot (35). Ultrastructural analysis by EM indicated that this enzyme had the typical barrel-shaped morphology described for MCP (36).
Purification and characterisation of glutathione reductase from scorpionfish (scorpaena porcus) and investigation of heavy metal ions inhibition
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2023
The purification process began with the homogenate preparation step. Ammonium sulphate precipitations in the range of 0–100% were performed on the prepared liver tissue. The GR enzyme precipitated in the intervals of 60–80% during the precipitation procedure. After precipitation, dialysis was performed to remove ions in the medium before affinity chromatography. Following dialysis, purification was performed on a 2′,5′-ADP Sepharose 4B affinity column, and the molecular weight of GR was determined using the SDS polyacrylamide gel electrophoresis. The liver tissue was purified 25.96 times with a yield of 28.277% and its molecular weight was determined to be 25 kDa. Quantitative protein determination was determined by the Bradford method. Ni2+, Mn2+, Cr3+, and Cd2+ heavy metals were applied on the purified enzyme. IC50 values of heavy metals were calculated as 2.4 µM, 30 µM, 135 µM and 206 µM, for Mn2+, Cd2+, Ni2+, and Cr3+, respectively.
An oxidation resistant pediocin PA-1 derivative and penocin A display effective anti-Listeria activity in a model human gut environment
Published in Gut Microbes, 2022
Taís M. Kuniyoshi, Paula M. O’Connor, Elaine Lawton, Dinesh Thapa, Beatriz Mesa-Pereira, Sara Abulu, Colin Hill, R. Paul Ross, Ricardo P. S. Oliveira, Paul D. Cotter
Pediocin PA-1 and pediocin M31L were purified as follows: 800 mL of P. acidilactici LMG 2351, a natural pediocin PA-1 producer,45 or E. coli/pETcoco-pedM31L cells were harvested by centrifugation at 10,000 x g for 15 min at 10 °C and the supernatant loaded onto an Econo column containing 60 g Amberlite XAD16N beads (Phenomenex, Macclesfield, CH, UK) prewashed with Milli Q water. The column was washed with 250 ml 30% ethanol and antimicrobial activity eluted with 250 ml 70% propan-2-ol (IPA) 0.1% TFA. To perform the ion exchange purification step, the IPA was removed from the eluent sample via rotary evaporation and the pH adjusted to approximately 4.4. The resulting sample was applied to an Econo column containing 60 ml SP-Sepharose beads (GE Healthcare, Bio-sciences AB, Uppsala, UP, SWE) pre-equilibrated with 250 ml 20 mM sodium acetate pH 4.4. The column was washed with 60 ml of 20 mM sodium acetate pH 4.4 and antimicrobial activity eluted with 250–300 ml 20 mM sodium acetate pH 4.4 containing 1 M NaCl. The salt-containing eluent from the SP-sepharose column was applied to a Strata–E C18 SPE column (Phenomenex) pre-equilibrated with methanol and water. The column was washed with 25% ethanol and antimicrobial activity eluted with IPA.
Enhancement of tumor immunogenicity by the introduction of non- proteinogenic amino acid azetidine-2-carboxylic acid
Published in OncoImmunology, 2022
Siyu Li, Shiqing Wang, Baorui Tian, Na Li, Yanan Chen, Yanhua Liu, Weijun Su, Yan Fan, Yongjun Piao, Jia Li, Longlong Wang, Jin Zhao, Shu Wang, Yi Shi, Rong Xiang
For the mass spectrometry analysis of GST proteins, 4T1 cells were treated with 0.5 mM Aze for 24 h. Total proteins of Aze-treated 4T1 cells were extracted using the cell lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, and protease inhibitor cocktail) and incubated with 50 μL of glutathione sepharose (Thermo Scientific, USA) at 4°C for 12 h. The sepharose resins were collected by centrifugation and washed with the wash buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM of EDTA, 0.1% Triton X-100) 3 times. Then the precipitates were resuspended in 50 μL 2× SDS sample buffer (Beyotime, Shanghai, China) and heated at 95°C for 5 min to elute the GST proteins for SDS-PAGE analysis. After coomassie-blue staining, the GST protein bands were cut for mass spectrum analysis (Biotech-pack, Beijing, China).