In this undergraduate biochemistry laboratory experiment, the vacuolar ATPase protein complex is purified from yeast cell extracts by doing immunoprecipitations under nondenaturing conditions. Immunoprecipitations are performed using monoclonal antibodies to facilitate data interpretation, and subunits are separated on the basis of their molecular size by SDS-PAGE. Both integral membrane and peripheral subunits are detected in Coomassie Blue-stained gels, and the identity of several subunits are confirmed by Western blots. Western blots serve to illustrate reproducibility and authenticity of the results and help students understand that they could use immunoblotting to detect subunits that are not visualized by Coomassie stain. The molecular mass of the subunits is estimated by their migration relative to molecular marker proteins in the same gel and used to estimate the size of the entire complex. Just like in biochemistry research, students learn to integrate several techniques to solve the subunit composition of a protein complex. These experiments are an alternative to classical protein purification techniques and can be adapted to study most protein complexes composed of membrane-bound and peripheral subunits from yeast. Instructors can use this approach to teaching proteomics and introduce students to systems biology. Because numerous yeast proteins are epitope tagged, instructors could use commercially available antibodies to study a protein complex of interest.Keywords: Immunoprecipitations, protein complexes, Western blots, protein purification, proteomics.Characterization of membrane-bound protein complexes is fundamental in biochemistry. Many cellular proteins are membrane-bound and possess multiple subunits that associate in a specific manner to enhance efficiency. However, isolation and characterization of membranebound proteins is a difficult task, which coupled with the complexity intrinsic with multiple subunits makes the study of most protein complexes a challenge.Immunoprecipitations offer an alternative to classical chromatographic techniques and can be used to purify proteins by one fractionation step. Nondenaturing immunoprecipitations, which are done under relatively mild conditions, can reveal components of steady-state assembled protein complexes [1, 2] as well as transient protein complexes [1]. Therefore, the protocol we describe can be further extended to introduce students to systems biology. The principle behind this powerful technique is simple. Antibodies are used to detect the protein under study as an antigen. Because antibodies have great affinity for their antigens, a highly specific antigen-antibody complex is formed. The antigen-antibody is complexed to the protein A-Sepharose (an antibody-binding protein linked to Sepharose beads) and harvested by centrifugation. Any protein(s) associated with the antigen is co-immunoprecipitated. SDS-PAGE is used to separate proteins on the basis of their molecular size, and standard protein markers in the same gel serve to generate calibra...
5-Diphenylphosphino-2-hydroxy-1,3-xylyl-18-crown-5 has been synthesized from 5-bromo-2-hydroxy-18-crown-5 by reacting it in sequence at low temperature with n-butyl lithium and methyl diphenylphosphonite. The phosphorous donor properties of this phenol phosphine (OH derivative) and the corresponding phenoxide (O -derivative) have been studied in the presence and absence of alkali metal ions by determining the frequencies of the A 1 (CO) bands of Ni(CO) 3 L complexes. For the OH and O -derivatives, the latter generated by addition of CsOH to the former, the (CO) bands are observed at 2067.6 and 2063.4 cm -1 , respectively, providing the trend predicted by Hammett parameters for OH and O -substituents. Addition of Na + or K + to the OH derivative has little effect on this stretching frequency, but the former ion shifts the O -derivative band to 2067.7 cm -1 . A solid state structure has been obtained of the OH derivative, and two independent molecules were found in the unit cell. Both have a single water molecule hydrogen bonded to two across-ring oxygen atoms and the phenol hydrogen. The crown ether ring has the usual gauche and anti arrangements for the C-C and C-O bonds.
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