Akta fplc system

Manufactured by Cytiva

Sourced in United States

The AKTA FPLC (Fast Protein Liquid Chromatography) system is a versatile laboratory instrument designed for protein purification and analysis. It is capable of performing a wide range of chromatographic techniques, including size exclusion, ion exchange, and affinity chromatography. The AKTA FPLC system is equipped with advanced software for precise control and monitoring of the purification process, allowing users to optimize their workflows and achieve consistent, high-quality results.

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Lab products found in correlation

Cmrl 1066 medium, by Thermo Fisher Scientific (2 mentions) Biomax 100 k concentrator, by Merck Group (2 mentions) Upc 900, by Cytiva (1 mentions) Deae sepharose fast flow column, by Cytiva (1 mentions) Phenyl sepharose, by Cytiva (1 mentions) Hybridoma sfm, by Thermo Fisher Scientific (1 mentions) Hiload 16 60 superdex 200 column, by GE Healthcare (1 mentions) Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Mono q 5 50 gl column, by Cytiva (1 mentions) Pierce bca kit, by Thermo Fisher Scientific (1 mentions) Mascot search engine, by Matrix Science (1 mentions) Amicon ultra filter, by Merck Group (1 mentions) Zetasize nano zs90, by Malvern Panalytical (1 mentions) Mouse elisa kit, by ALPCO (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Protein standard mix 15 600 kda, by Merck Group (1 mentions) Superdex 200 increase 10 300 gl column, by GE Healthcare (1 mentions) Pcrii vector, by Thermo Fisher Scientific (1 mentions) Superose 6 10 300 gl column, by Cytiva (1 mentions) Zetasizer nano zs90, by Malvern Panalytical (1 mentions)

32 protocols using akta fplc system

Purification of Proteasome from T. bernacchii

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The proteasome active fractions, recovered after each purification step, were detected by measuring the CT-like activity using the specific fluorogenic substrate LLVY. The T. bernacchii hemolysates were loaded on a DEAE Sepharose Fast Flow column, previously equilibrated in 25 mM Tris/HCl, pH 7.5 (Buffer A), and connected to an AKTA_FPLC system (Amersham Biosciences). Bound proteins were eluted using an ionic strength gradient from 0 to 1 M NaCl in Buffer A at a flow rate of 1 ml·min⁻¹. The active fractions were pooled, dialysed extensively against 25 mM Tris/HCl, pH 7.5 and 1 M ammonium sulfate (Buffer A) and then loaded on to a Phenyl Sepharose (Amersham) column, connected to an AKTA_FPLC system (Amersham Biosciences), equilibrated in the same buffer. Bound proteins were eluted with a linear gradient (0–100%) of 25 mM Tris/HCl (pH 7.5) (buffer B) at a flow rate of 1 ml·min⁻¹. The active fractions were pooled, dialysed against 25 mM Tris/HCl (pH 7.5) and then applied to a Superdex 200 PC 3.2/30 column connected to a SMART System (Pharmacia), equilibrated in 25 mM Tris/HCl, pH 7.5 and 50 mM NaCl. Finally, active fractions were pooled and the purified proteasome was stored in 25 mM Tris/HCl pH 7.5, containing 5% glycerol.

Gogliettino M., Balestrieri M., Riccio A., Facchiano A., Fusco C., Palazzo V.C., Rossi M., Cocca E, & Palmieri G. (2016). Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii. Bioscience Reports, 36(2), e00320.

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Oligomeric State Determination of GST-tagged Proteins

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Oligomeric state of the variants in GST‐tagged as well as untagged forms were determined using an Amersham‐Pharmacia Biotech AKTA‐FPLC system (Kwai Chung, Hong Kong) with Superdex‐200 HiLoad 16/600 or Superdex‐200 Increase 10/30 GL columns as described earlier 25. The molecular weight markers used were ovalbumin (44 kDa), bovine serum albumin (66 kDa), Yeno (93 kDa), alcohol dehydrogenase (150 kDa) and ß‐amylase (200 kDa).

Dutta S., Moitra A., Mukherjee D, & Jarori G.K. (2017). Functions of tryptophan residues in EWGWS insert of Plasmodium falciparum enolase. FEBS Open Bio, 7(7), 892-904.

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Oligomeric State Analysis of HIGLE Proteins by SEC

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Size exclusion chromatography (SEC) was performed to analyze the oligomeric state of At-HIGLE, At-HIGLE^1–183 and At-HIGLE^184-368 using the AKTA-FPLC system (Amersham). Sephacryl 16/60 S-100 (for At-HIGLE^1–183 and At-HIGLE^184–368) and Sephacryl 16/60 S-200 columns (for At-HIGLE) were equilibrated with 20 mM Tris–HCl (pH 8.5), 5% glycerol, 5 mM beta-mercaptoethanol and 0.5 M NaCl. The column was calibrated using gel filtration markers (Bio-RAD). A standard curve was generated using gel filtration markers (Vitamin B12, Myoglobin, Ovalbumin and gamma globulin). Kav was calculated as (Ve – Vo)/(Vt – Vo), where Ve, Vo and Vt are elution volume, void volume, and total volume, respectively. Thyroglobulin (670 kDa) was used to determine the column's void volume.

Verma P., Kumari P., Negi S., Yadav G, & Gaur V. (2022). Holliday junction resolution by At-HIGLE: an SLX1 lineage endonuclease from Arabidopsis thaliana with a novel in-built regulatory mechanism. Nucleic Acids Research, 50(8), 4630-4646.

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Production and Purification of Recombinant Human Troponin Complex

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Recombinant human troponin complex was produced as described in detail previously.^{5 (link)} Briefly, three different cTnI forms were made via site-directed mutations of Ser42/44 and Ser23/24 into aspartic acid (D) to mimic phosphorylation or into alanine (A) to mimic dephosphorylation: 42D/44D, 42A/44A and 23D/24D. cDNA encoding human cardiac isoforms (troponin C (cTnC), myc-tag labeled cTnT (cTnT-myc), cTnI wild-type, and cTnI mutants) were transformed in E. coli Rosetta2^{24 (link)} and cultured under carbenicillin/chloramphenicol selection in Overnight Express TB medium (EMD Biosciences). Cultures were harvested by centrifugation, re-suspended in phosphate buffered saline (PBS), and centrifuged at 10000xg. Pellets were stored at −80°C until used. Troponin subunits were purified using fast protein liquid chromatography (AKTA-FPLC System Amersham Biosciences) essentially as described previously.^{24 (link)}Fractions containing equal ratios of cTnT, cTnC and cTnI subunits were pooled, subsequently purified by AKTA-FPLC chromatography using a Resource Q to remove residual uncomplexed troponin subunit and finally dialyzed against 10 mM imidazole, 200 mM KCl, 5 mM MgCl₂, 2.5 mM EGTA, 1 mM DTT, 0.1 mM PMSF (pH 6.9; twice, 1L each) prior to concentrating the complexes to a final concentration >2 mg/mL by centrifugation using Centriprep YM-10 centrifugal filters (Millipore).

Wijnker P.J., Sequeira V., Witjas-Paalberends E.R., Foster D.B., dos Remedios C.G., Murphy A.M., Stienen G.J, & van der Velden J. (2014). Phosphorylation of protein kinase C sites Ser42/44 decreases Ca2+-sensitivity and blunts enhanced length-dependent activation in response to protein kinase A in human cardiomyocytes. Archives of biochemistry and biophysics, 554, 11-21.

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Dynamic Perifusion of Isolated Islets

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Isolated islets (from the same groups as above) recovered overnight in CMRL-1066 medium (Gibco) containing 10% fetal bovine serum and 2 mmol/l L-glutamine at 37 °C. Thirty islets per NHP were placed in a dynamic perifusion system (Amersham Biosciences AKTA FPLC System) as previously described^{37 (link)}. To summarize, the perifusion was performed using Krebs buffer with 2.8 mM glucose at a flow rate of 1 ml/min for 30 min to establish stable basal insulin secretion. Next, the islets were perifused with 2.8 mM glucose for 10 min, and fractions of 500 μl were collected every 30 s. Then, the glucose concentration was increased to 20 mM, and fractions of 500 μl were collected every 30 s for 20 min. Finally, the islets were perifused with 30 mM KCl, and fractions of 500 μl were collected every 30 s for 10 min. After the perifusion, the islets were recollected from the column for protein quantification. The insulin in the effluent was measured as described above. The fractional insulin secretion rate was calculated as secreted insulin per minute normalized to the protein content.

Kalsi R.S., Kreger A.M., Saleh M., Yoshida S., Sharma K., Fusco J., Saloman J.L., Zhang T., Thomas M., Sehrawat A., Wang Y., Reif J., Mills J., Raad S., Zengin B., Gomez A., Singhi A., Tadros S., Slivka A., Esni F., Prasadan K, & Gittes G. (2023). Chemical pancreatectomy in non-human primates ablates the acini and ducts and enhances beta-cell function. Scientific Reports, 13, 9113.

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Purification of Target Protein Using IMAC

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For AviPure purification, 5 g biomass (pellets) from the previously mini-pilot scale fermentation was re-suspended in 40 mL sonication/Wash buffer (50 mM Phosphate Buffer pH 7.5, 300 mM NaCl, 10 mM Imidazole) and 350 µL protease inhibitor cocktail mix. Direct sonication was performed for 45 min using amplitude of 90 % and 0.6 cycles; with the beaker containing cell suspension placed in ice/ice cold water and was continuously stirred to keep cool. After sonication, the lysate was centrifuged for 1 h at 16,000 rpm using a Beckman Coulot Avanti J-E centrifuge to get rid of cell debris. IMAC was used to purify the target protein and a 5 mL Ni–NTA column volume was chosen, equilibrated with sonication/wash buffer before loading. After centrifugation, the final 40 mL supernatant was loaded on the column. UPC–900 Amersham Biosciences AKTA FPLC system was employed for the purification. The elution buffer used was the same as the sonication/wash buffer, but contained a higher imidazole concentration (250 mM). The obtained protein was analyzed by SDS-PAGE following the method of Laemmli.

Kangwa M., Yelemane V., Polat A.N., Gorrepati K.D., Grasselli M, & Fernández-Lahore M. (2015). High-level fed-batch fermentative expression of an engineered Staphylococcal protein A based ligand in E. coli: purification and characterization. AMB Express, 5, 70.

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Ion Exchange Chromatography of S. chromogenes

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Twenty milliliter of S. chromogenes <3-kDa supernatant was loaded on a Sigma-Aldrich HiTrap^® SP Fast Flow column on an Amersham Pharmacia Biotech AKTA FPLC system. Samples were eluted in a 0–50% gradient of solvent B (50-mM Tris pH 8.0) over solvent A (20-mM Tris pH 7.5). Five milliliter fractions were collected at a flow rate of 2.0 mL/min.

Chin D., Goncheva M.I., Flannagan R.S., Deecker S.R., Guariglia-Oropeza V., Ensminger A.W, & Heinrichs D.E. (2021). Coagulase-negative staphylococci release a purine analog that inhibits Staphylococcus aureus virulence. Nature Communications, 12, 1887.

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Biotinylated Bait Saturation of AF488-Streptavidin

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To determine how much biotinylated bait was needed to saturate a fixed amount of AF488-streptavidin (Thermo Fisher Scientific, cat. no. S11223), different amounts of biotinylated bait proteins were incubated with AF488-streptavidin at 4°C overnight, and then complexes were purified by size exclusion chromatography (SEC) using an Amersham Biosciences AKTA FPLC System (Amersham). Fractions with the largest size, representing the largest complex with the most bait proteins, were collected and used for cell staining and sorting.

Yang L., Sheets T.P., Feng Y., Yu G., Bajgain P., Hsu K.S., So D., Seaman S., Lee J., Lin L., Evans C.N., Guest M.R., Chari R, & St. Croix B. (2024). Uncovering receptor-ligand interactions using a high-avidity CRISPR activation screening platform. Science Advances, 10(7), eadj2445.

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Islet Perifusion Protocol for Insulin Secretion

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Isolated islets (from the same groups as above) recovered overnight in CMRL-1066 medium (Gibco) containing 10% fetal bovine serum and 2 mmol/l L-glutamine at 37°C. Thirty islets per NHP were placed in a dynamic perifusion system (Amersham Biosciences AKTA FPLC System) as previously described^{33 (link)}. To summarize, the perifusion was performed using Krebs buffer with 2.8 mM glucose at a flow rate of 1 ml/min for 30 minutes to establish stable basal insulin secretion. Next, the islets were perifused with 2.8 mM glucose for 10 minutes, and fractions of 500 μl were collected every 30 seconds. Then, the glucose concentration was increased to 20 mM, and fractions of 500 μl were collected every 30 seconds for 20 minutes. Finally, the islets were perifused with 30 mM KCl, and fractions of 500 μl were collected every 30 seconds for 10 minutes. After the perifusion, the islets were recollected from the column for protein quantification. The insulin in the effluent was measured as described above. The fractional insulin secretion rate was calculated as secreted insulin per minute normalized to the protein content.

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Gel Filtration Chromatography of Bacterial Regulators

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Gel filtration chromatography was carried out on a Hiload 16/60 Superdex 75 pg (Amersham Biosciences) and Superose^® 12 10/300 GL (Sigma-Aldrich) size exclusion chromatography columns for OmpR and EnvZc, respectively, on an AKTA FPLC system (Amersham Biosciences) using suitable buffers. Two column volumes of running buffer was used to pre-equilibrate, the flow rate was 0.5 ml min⁻¹. Eluted peaks were quantified by absorbance at 280 nm and fractions were analyzed by SDS-PAGE.

Chakraborty S., Winardhi R.S., Morgan L.K., Yan J, & Kenney L.J. (2017). Non-canonical activation of OmpR drives acid and osmotic stress responses in single bacterial cells. Nature Communications, 8, 1587.

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