Selective Detection Of Proteins Research Articles

Surface plasmon resonance detection for capillary electrophoresis separations.

A miniaturized surface plasmon resonance sensor has been used as an on-line detector for capillary electrophoresis separations. The capillary was modified slightly to shield the sensor electronics from the high voltages applied during the separation. A three-component mixture of high refractive index materials was separated and detected at the millimolar level by an untreated gold-sensing surface. A simple protein immobilization procedure was used to functionalize the surface for selective protein detection. A hybrid buffer system was developed, in which both the deposition of immobilized protein layers and the electrophoretic delivery of protein analytes were optimized. The detection system has a reproducibility of 15%, a dynamic range of 3 orders of magnitude, and a detection limit for IgG of 2 fmol.

Selective Detection of Membrane Proteins Without Antibodies: A Mass Spectrometric Version of the Western Blot

A method has been developed, called the mass western experiment in analogy to the Western blot, to detect the presence of specific proteins in complex mixtures without the need for antibodies. Proteins are identified with high sensitivity and selectivity, and their abundances are compared between samples. Membrane protein extracts were labeled with custom isotope-coded affinity tag reagents and digested, and the labeled peptides were analyzed by liquid chromatography-tandem mass spectrometry. Ions corresponding to anticipated tryptic peptides from the proteins of interest were continuously subjected to collision-induced dissociation in an ion trap mass spectrometer; heavy and light isotope-coded affinity tag-labeled peptides were simultaneously trapped and fragmented accomplishing identification and quantitation in a single mass spectrum. This application of ion trap selective reaction monitoring maximizes sensitivity, enabling analysis of peptides that would otherwise go undetected. The cell surface proteins prostate stem cell antigen (PSCA) and ErbB2 were detected in prostate and breast tumor cell lines in which they are expressed in known abundances spanning orders of magnitude.

Expressions of P-selectin- and HSP72-like immunoreactivities in rat brain after transient middle cerebral artery occlusion

The role of an adhesion molecule such as P-selectin may be important in the pathogenesis of stroke. However, temporal, spatial and cellular profiles of the expression of such a protein have not been fully studied. Change of immunoreactive P-selectin was examined in rat brain after transient middle cerebral artery (MCA) occlusion in comparison with that of 72 kDa heat shock protein (HSP72) which is a well known marker of cell injury. Western blot analyses were performed to ensure the selective detection of immunoreactive P-selectin and HSP72 proteins with each antibody using brain samples before and after ischemia. Temporal, spatial and cellular changes of immunohistochemical expressions of P-selectin and HSP72 were evaluated with rat brain sections at 2 and 8 h, and 1, 3 and 7 days of reperfusion after 1 h of MCA occlusion (MCAO). Hematoxylin-eosin (HE) staining was performed to evaluate brain cell damage at 3 and 7 days of reperfusion. Western blot showed a single band at molecular weights of 140 and 72 kDa for P-selectin and HSP72, respectively, only after ischemia. No significant band was observed without primary antibody. P-selectin-like immunoreactivity was not normally present in rat brain sections. However, it was expressed mainly in the post-capillary venules of the cerebral cortex and caudate in the MCA territory with a peak at 8 h to 1 day. The expression was diminished by 3 days of reperfusion. An immunoreactive HSP72 was scarcely present in the cerebral cortex and caudate of the sham control brain. However, the protein was induced in neurons of the MCA territory. The HSP72 induction was gradually intensified from 8 h with peaks at 1 day in the cortex and at 3 days in the caudate. The immunoreactivity decreased by 7 days. Histopathological study with HE staining showed no evident cell damage at 3 and 7 days of reperfusion. The present results indicate that temporal, spatial and cellular differences were present in the expressions of immunoreactive P-selectin and HSP72 proteins. P-selectin was expressed from an earlier stage of reperfusion in post-capillary venules, and the expression became maximum at the same time both in the cerebral cortex and caudate. In contrast, HSP72 induction began later in neurons and reached maximum at a different time between the cortex and caudate.

Selective silver-staining methods for RNA and proteins in the same polyacrylamide gels

Two silver-staining methods for selective and ultrasensitive detection of RNAs and proteins in the same polyacrylamide gels were developed, both derived from procedures recommended for protein staining. The first, a double-staining technic with Coomassie brilliant blue and ammoniacal silver, allows visualization of RNAs as negative bands and proteins as dark brown bands. The second is also a double-staining technique, but uses silver in both steps. This second method develops the RNA bands first and then the protein bands. These techniques, especially the second, permit characterization of the different components of ribonucleoproteic complexes in the same electrophoresis gels.

High-voltage isoelectric focusing in ultrathin gels and enzyme-amplified immunoassay: a new method for analysis of cerebrospinal fluid proteins.

A procedure using high-voltage isoelectric focusing (IF) in ultrathin (02. mm) gels and enzyme-amplified immuno-sandwich assay was elaborated to get optimal IF separation conditions, to avoid CSF concentration, e.g. by ultrafiltration preceding IF with the risk of unequal protein losses, to minimize the amounts of CSF and expensive reagents needed, especially antibodies and to shorten the analysis time, including the selective detection of proteins. The high voltage (2000-3000 V/10 cm) and efficient cooling during IF were obtained using ECPS 3000/150 and FBE 3000 (Pharmacia, Sweden). Ampholytes (Pharmalytes) of different pI intervals were used. The CSF and (diluted) serum samples were microdialysed in polyacrylamide gel before IF to minimize band curvature and to obtain optimal resolution. The IF separation was performed in about 1 h. Owing to the rapid fixation of ultrathin gels after IF, full use could be made of the high-voltage resolving capacity. The thin gels also made histochemical techniques applicable. Different immunological identification assays have been tested. An enzyme-amplified (alkaline phosphatase) immuno-sandwich method was found to be very sensitive and selective, and has so far given the best results. Many proteins in the same sample, applied as a line on the gel before IF, could be detected by overlaying antibody-soaked membrane strips. Furthermore, one specific protein could be examined in many samples simultaneously by overlaying or immersion of diluted antibody solutions. A few microlitres of unconcentrated CSF and diluted serum were used for the analysis performed within 1 day. The findings for albumin, transferrin and IgG in CSF and sera from patients with different neurological diseases, especially including cases with "normal" CSF, barrier damage, degenerative and demyelinating disorders, have been compared with the corresponding protein-stained (Coomassie R-250) patterns where the CSF had been concentrated by a special vacuum evaporation technique before IF.

Changing patterns of single-stranded-DNA-binding proteins in differentiating brain cortex and cerebellar neurons.

Single-stranded-DNA-binding proteins were analyzed in nuclei of differentiating rat cortex and cerebellar neurons. The developmental period investigated ranged from gestational day 19 (i.e. 3 days before term) to postnatal day 30. During this time both types of neurons undergo transition from proliferating, undifferentiated precursor cells to non-proliferating, terminally differentiated neurons. For comparison, nuclei from mature cortex glia and liver were also examined. Nuclei were isolated according to cell type, the proteins were 14C-labeled in vitro by reductive methylation and were fractionated by affinity chromatography on tandemly arranged columns of double-stranded and single-stranded DNA-cellulose. The columns were uncoupled and the proteins adsorbed to the single-stranded DNA were eluted with salt. They were then analyzed by high resolution two-dimensional gel electrophoresis followed by fluorography. This strategy ensured the selective detection of proteins that recognize single-stranded DNA specifically, and eliminated interference by proteins binding to DNA by simple ionic interaction as well as by proteins with affinity for double-stranded DNA. Many single-stranded-DNA-binding proteins showed conspicuous developmental fluctuations. In cortex neurons these took place around the time of birth and the first postnatal week, whereas in cerebellar neurons they occurred later and in a more protracted fashion. Thus, in both cortex and cerebellar neurons the protein changes followed a time course closely paralleling the arrest of cell division and the beginning of terminal differentiation. It is suggested that this approach may lead to the detection of putative regulatory proteins of the cell nucleus.