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MBP Purification Protocols

MBP or maltose binding protein is found in the bacteria Escherichia coli, which is part of the gut flora of humans and animals. The purification of MBP involves a series of procedures aimed at separating the protein's constituents. In molecular biology, MBP purification is a vital process for the study of the structure and function of this protein. MBP purification protocols include MBP fused proteins purification, small scale MBP-fusion and maltose binding protein fusion tag purification.

  1. MBP Fused Proteins Purification

    • During this protocol, technicians wash 20 ml of the polymer amylose resin with aTris solution at pH 7.4, sodium chloride, Mercaptoethanol (ME) and ethylenediaminetetraacetic acid (EDTA), according to Protocols Online. Cell samples are added, centrifuged and analyzed on gel electrophoresis. Samples are also analyzed in a chromatographer, where technicians eluate or observe the solution's constituents while passing through the chromatography column.

    Small Scale MBP-fusion Protein Purification

    • According to the Hebrew University of Jerusalem, biologists use the reagent isopropyl b-D-1-thiogalactopyranoside (IPTG), spin cell culture for 10 min at 8,000 rotations per minute at 39.2 degrees Fahrenheit. Later, they remove supernatant liquid and mix cold PBS buffer to the sample, which is divided into several tubes and centrifuged again. Samples are then kept at -112 degrees Fahrenheit for posterior analysis.

    Maltose Binding Protein Fusion Tag Purification

    • Affinity tags are genetically engineered molecules employed in molecular biology to detect and purify proteins. This protocol is a complex set of procedures divided into three stages, which include the construction of polyhistidine protein marker (HisMBP); a pilot experiment to assess the solubility of the protein; and the large-scale purification of MBP molecules, according to Nature Protocols. Apart from E.coli samples, technicians use ampicillin, kanamycin, IPTG, anhydrotetracycline, glucose monohydrate, cell lysis buffer, DNA polymerase, PCR primers, Tris-acetate-EDTA and ethidium bromide.

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