By Senta Reichelt (eds.)
The goal of this variation is to introduce the newbie to the fundamentals of affinity chromatography and supply functional wisdom for the advance of affinity separation protocols. Affinity Chromatography: equipment and Protocols, 3rd Edition publications readers via new cutting-edge protocols, molecular modelling, and the examine of ligand-target interactions. Written within the winning Methods in Molecular Biology sequence structure, chapters contain introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, simply reproducible protocols, and notes on troubleshooting and warding off recognized pitfalls.
Authoritative and simply accessible, Affinity Chromatography: tools and Protocols, 3rd Edition is designed as an invaluable source for these drawn to the swift and quantitative isolation of biomolecules with excessive purity.
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Additional info for Affinity Chromatography: Methods and Protocols
Dilute 100 mL of 10Â running buffer with 890 mL of water, mix well, and add 10 mL of 10 % SDS solution. Care should be taken to add the SDS solution last and to mix gently, since it makes bubbles. 6. This method is prepared to purify PAT protein from 100 mL of E. coli cell culture. One gram E. coli cell paste can produce up to 10 mg PAT protein. 6 mg PAT protein. You can proportionally scale up or down according to the amount of cell extract/lysate you start with. 7. This chromatography can easily be conducted using gravity flow.
Load 10 μl of each sample on SDS-PAGE gel (Fig. 2). Fig. 2 SDS-PAGE of PAT purified from transgenic cottonseed using Reactive brown 10-agarose. Proteins were separated by SDS-PAGE on a 4–20 % (w/v) Tris glycine gel and stained with InstantBlue. Lane 1: molecular weight markers (Bench Mark prestained, Invitrogen, Carlsbad, CA), Lane 2: an E. coli-produced PAT protein standard, 3 μg, Lane 3: Transgenic cottonseed extracts, Lane 4: Transgenic cottonseed extracts after treated with 10 mM CaCl2, Lane 5: Reactive brown 10 flow through, Lane 6: Elution from Reactive brown 10.
Incubate the agarose in elution buffer for at least 10 min at 4 C. 9. Remove the column’s plug and cap. Allow the PAT-containing elution to drain into a collection tube. Store the elution at 4 C. Save a small sample for SDS-PAGE analysis. 10. Analyze collected fractions from above steps by SDS-PAGE (see Note 9). Load 10 μl of each sample on SDS-PAGE gel (Fig. 1). Fig. 1 SDS-PAGE of PAT purified from E. coli lysate using Reactive brown 10-agarose. Proteins were separated by SDS-PAGE on a 4–20 % (w/v) Tris glycine gel and stained with InstantBlue.