Gel Staining Techniques Research Articles

Protein‐Protein Interactions Identified by Pull‐Down Experiments and Mass Spectrometry

The aim of this unit is to provide a method for the identification of new protein-protein interactions. Pull-down experiments with GST fusion proteins attached to glutathione beads are a screening technique for identification of protein-protein interactions. When coupled with mass spectrometry, pull-downs can be considered as the protein-based equivalent of a yeast two-hybrid screen. To improve the isolation of specific binding partners, pull-down methods are described involving the use of cross-linking, large-scale tissue lysates, and spin columns. Alternative techniques are detailed for isolating activation state-dependent protein interactions with small GTPases. Appropriate methods of sample preparation for mass spectrometry-based identification of interacting proteins are described, including specialized gel staining techniques, band excision, and in-gel tryptic digestion. Data interpretation and the most commonly encountered problems are discussed.

Development of gel staining techniques for detecting the secretion of procathepsin D (52-kDa protein) in MCF-7 human breast cancer cells

17β-Estradiol stimulates the secretion of the 34- and 52-kDa protein (i.e., cathepsin D and procathepsin D, respectively) from MCF-7 human breast cancer cells and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) inhibits this estrogen-stimulated response. A comparison of the effects of 17β-estradiol, TCDD, and their combinations on the secretion of these two proteins was determined using four different assay procedures, namely autoradiographic analysis of the 35S-labeled proteins (from [ 35S]methionine) separated by polyacrylamide gel electrophoresis (PAGE), densitometric analysis of the silver- and double-stained proteins separated by PAGE, and radioimmunoassay of the proteins using commercially available antibodies to the 52-kDa protein. The results showed that the autoradiographic, staining, and radioimmunoassay procedures gave comparable results with only a few minor differences in the relative amounts of the 52-kDa detected in the various treatment groups. In the medium obtained from 17β-estradiol-treated cells that was serially diluted, there was an excellent linear correlation for the relative concentrations of the 52-kDa protein using the double-staining/densitometric procedure and the radioimmunoassay. These results indicate that the double- or silver-staining method may be a useful and rapid method for screening new compounds as antiestrogens in MCF-7 cells.

Bifunctional Protein in Carrot Contains Both Aspartokinase and Homoserine Dehydrogenase Activities

We have purified homoserine dehydrogenase to homogeneity and subjected polypeptide fragments derived from digests of the protein to amino acid sequencing. The amino acid sequence of homoserine dehydrogenase from carrot (Daucus carota) indicates that in carrot both aspartokinase and homoserine dehydrogenase activities reside on the same protein. Additional evidence that aspartokinase and homoserine dehydrogenase reside on a bifunctional protein is provided by coelution of activities during purification steps and by enzyme-specific gel staining techniques. Highly purified fractions containing aspartokinase activity were stained for aspartokinase activity, homoserine dehydrogenase activity, and protein. These gels confirmed that aspartokinase activity and homoserine dehydrogenase activity were present on the same protein. This arrangement of aspartokinase and homoserine dehydrogenase activities residing on the same protein is also found in Escherichia coli, which has two bifunctional enzymes, aspartokinase I-homoserine dehydrogenase I and aspartokinase II-homoserine dehydrogenase II. The amino acid sequence of the major form of homoserine dehydrogenase from carrot cell suspension cultures most closely resembles that of the E. coli ThrA gene product aspartokinase I-homoserine dehydrogenase I.

Microtubule-associated proteins: a monoclonal antibody to MAP2 binds to differentiated neurons.

Hybridomas that secret IgG reacting specifically with the brain microtubule-associated protein MAP2 have been prepared with speen cells from BALB/c mice hyperimmunized with high molecular weight neurotubule-associated proteins. Immunofluorecence microscopy using dual fluorochrome labeling of tubulin and MAP2 antigens revealed identical patterns of interphase fiber networks in cells from explants of newborn mouse brain. The anti-MAP2 antibody did not stain primary mouse kidney cells or CHO, 3T3, HeLa, or PtK1 cell lines. Immunoprecipitation and antibody gel staining techniques failed to demonstrate any crossreacting antigen in these cells. MAP2 antigen was not seen in association with the mitotic spindle in any of the cells examined. Radioimmunoassay showed species crossreactivity of the anti-MAP2 antibody with mammalian but not avian neural cell extracts. Glial cells and some neuroblastoma cell lines did not appear to contain MAP2. However, in the B104 rat neuroblastoma cell line the MAP2 antigen appeared to be associated with the cytoskeleton concomitant with differentiation induced by dibutyryl cyclic AMP. In disagreement with most previously published reports, our data suggest that MAP2 is found only in differentiated neuronal cells and raises the possibility that MAP2 is involved in neuronal differentiation or neuron-specific processes.

Internal Standards for Molecular Weight Determinations of Proteins by Polyacrylamide Gel Electrophoresis: APPLICATIONS TO ENVELOPE PROTEINS OF ESCHERICHIA COLI

Abstract A new technique was devised to be used with molecular weight determinations of proteins on polyacrylamide gels. Internal standards consisting of proteins covalently linked to 1-dimethylaminonaphthalene-5-sulfonyl- (DANS-) groups were prepared. DANS- derivatives of insulin and monomer, dimer, trimer, tetramer, pentamer, and hexamer of egg white lysozyme and serum albumin covered molecular weights from 6,600 to 360,000. They were concurrently applied in small amounts on a single gel together with the unknown sample. Proteins were separated by electrophoresis in 0.5% sodium dodecyl sulfate. Positions of the DANS- proteins were detected under an ultraviolet lamp as yellowish fluorescent bands. These bands are reproducible and related to the positions obtained by standard gel staining techniques. Even 0.2 µg of a DANS-protein was detectable. Molecular weights of unknowns were determined by interpolation from their positions. This method was applied to envelope proteins of Escherichia coli. The molecular weights of major proteins were determined. The molecular weights of Proteins X and Y, which have been shown in a previous paper to be related to cell division and DNA synthesis, respectively, were reexamined and found to be 50,000 and 44,000, respectively. One of the major envelope proteins was found at the molecular weight of 7500. This protein showed a very high arginine to histidine ratio (about 11 times as high as the others) distinctly different from all other proteins of higher molecular weights. Thus, this protein is not a subunit of proteins of higher molecular weights.