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At least 523 records · Page 29Linked to original sources
PubMed1993

Covalent attachment of peptides to membranes for dot-blot analysis of glycosylation sites and epitopes.

Noncovalent binding of proteins to membranes is often employed for dot-blot analysis with various visualization techniques. These techniques are usually not applicable to peptide dot-blot analysis due to peptide wash-off during the staining procedure. As exemplified with a synthetic peptide and peptides produced by proteolysis of a protein, it is possible to achieve efficient covalent attachment to Immobilon-AV membranes. The utility of this membrane has been demonstrated with immunostaining and carbohydrate staining procedures.

Amino Acid Sequence↗
PubMed1993

A cDNA cloning method for monomeric GTP-binding proteins by ligand blotting.

We have developed a convenient method of cDNA cloning for monomeric GTP-binding proteins. The method consists of a serial dilution of cDNA expression library, a separation of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and a ligand blotting; each group containing the proteins from about 1000 independent colonies was separated by SDS-PAGE, transferred to nitrocellulose filters, incubated with 10(-9) M [alpha-32P]GTP, and autoradiographed. Separation of proteins by electrophoresis resulted in a drastic reduction of nonspecific binding of labeled GTP to the filters. A serial dilution of colonies resulted in a single clone having GTP-binding activity. We checked about 60,000 colonies and detected two signals. This method is dependent on the ligand specificity of the expressed proteins and is different from the conventional methods based on DNA sequence similarity. This method can be used to isolate cDNAs encoding novel GTP-binding proteins.

Bacteriophage lambda↗
PubMed1993

Detection of iron binding proteins by a blotting technique.

We describe a simple method for detecting iron binding proteins based on binding of 59Fe to protein blots from SDS gels followed by autoradiography. This binding is specific and is not affected by other metal ions. The method has allowed identification of new iron binding proteins in intestinal mucosa and in Hela cells.

Animals↗
PubMed1993

A semidry electroblotting system efficiently transfers both high- and low-molecular-weight proteins separated by SDS-PAGE.

A general procedure is described for the simultaneous recovery by electroblotting on nitrocellulose or polyvinylidene difluoride (PVDF) membranes of both high- and low-molecular-weight proteins already separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is based on the use of an original set of buffers which creates a stable pH boundary between the two faces of the blotted membrane. The gel to be transferred is placed on the basic side (pH 8.4) of this boundary, which also contains sodium dodecyl sulfate. Under these conditions even the high-molecular-weight proteins are efficiently eluted out of the acrylamide. Their migration is then stopped on the membrane, due to the change of pH with the acid side (pH 3.8) of the boundary, and the presence of methanol in it. This increases the adsorption on the membrane of the low-molecular-weight polypeptides which have a low affinity for nitrocellulose or PVDF. The procedure permits a quantitative transfer of complex samples which contain poorly soluble proteins or with molecular masses outside the range of 20-70 kDa, like grain storage proteins of cereals.

Buffers↗
PubMed1993

Ligand blot analysis: validation of quantitative capabilities and utilization for measurement of truncated insulin-like growth factor regulation of Hep-G2 insulin-like growth factor binding protein-1 production.

The quantitative capabilities of Western "ligand" blot analysis (LBA) of insulin-like growth factor binding protein have been evaluated using iodine-labeled insulin-like growth factor-II (125I-IGF-II), purified amniotic fluid insulin-like growth factor binding protein-1 (IGFBP-1), and IGFBP-1 in conditioned medium (CM) from Hep-G2 cells. Optimal conditions were evaluated for analysis of IGFBP-1 using IGF-2-II as the ligand. IGFBP-1 ligand blot quantities were measured after autoradiography by one- (1D) and two (2D)-dimensional densitometry. The 2D method was found to exhibit higher linearity (IGF-I, r = 0.993; IGF-II, r = 0.984) over a wider range than the 1D method. Displacement curves for IGF-II against membrane-bound IGFBP-1 measured by LBA were similar to those obtained with IGFBP-1 solutions using antibody precipitation of IGFBP-1. Transfer of IGFBP-1 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels to nitrocellulose membranes was linear across the range 3.1-400 ng with both the 0.45- and the 0.2-micron pore sizes tested. The 0.2-micron size, however, showed nearly quantitative binding of IGFBP-1 trapping 20% more of this binding protein than the larger pore size. LBA was used to measure IGFBP-1 in CM from control and des1-3IGF-I (trIGF-I)-treated Hep-G2 cells and the results were compared with those obtained by a specific IGFBP-1 radioimmunoassay. IGFBP-1 concentrations measured by both assays were similar and both demonstrated nearly identical sensitivities. Each assay confirmed the previously noted trIGF-I suppression of IGFBP-1 secretion by Hep-G2 cells.

Animals↗
PubMed1993

Cloning of a cDNA encoding an RNA binding protein by screening expression libraries using a northwestern strategy.

Successful cloning with cDNA expression libraries involves interaction of ligand molecules (probes) with expressed fusion proteins of interest. So far those ligands leading to successful results fall into three classes: (i) antibodies, (ii) protein ligands that interact with the protein of interest, and (iii) DNA sequences recognized by transcription factors. We have previously identified a 50-kDa protein (called DSEF-1) which interacts with a functionally important 14-base G-rich RNA sequence located downstream of the simian virus 40 late polyadenylation signal. By using small RNAs containing the DSEF-1 binding site as probes, a cDNA clone was isolated whose gene product interacted in a sequence-specific fashion with the DSEF-1 binding site. This RNA binding protein contains three potential RNA recognition motifs. We present here a procedure to obtain cDNA clones of RNA binding proteins using recognition site probes.

Bacteriophage lambda↗
PubMed1993

Secretory cell immunoscreening assay--a highly sensitive screening method for secretory cells.

We have developed a rapid and sensitive screening method to detect the secretory product from transformed secretory cells. Polyclonal antibodies against the target secretory protein are first adsorbed onto a nitrocellulose membrane and are then called solid-phase antibodies. Then this membrane is placed on top of target protein-secreting adherent animal cell colonies. After trapping the secreted protein on the membrane, the protein is detected using the highly sensitive enhanced chemiluminescence detection system. Thus, this method is named the secretory cell immunoscreening assay (SCISA). We have screened recombinant BHK-21 cells, which secrete human interleukin-6 (hIL-6) for highly productive clones. With the SCISA method, it is possible to detect less than 100 pg of hIL-6 secreted by a single colony. We are also able to select highly productive colonies rapidly and easily from a large sample population of transformed cell colonies.

Animals↗
PubMed1994

A dot-blot assay of metabolically radiolabeled hyaluronan.

Glycosaminoglycans labeled in tissue culture in the presence of [3H]glucosamine were proteolytically solubilized and then precipitated with cetylpyridinium chloride onto a sheet of nitrocellulose using a dot-blot apparatus. The proportion of hyaluronan (HA) was calculated from parallel aliquots digested with Streptomyces hyaluronidase, an enzyme specifically degrading HA. A linear response of radioactivity was obtained for samples in the range of 50-10,000 cpm (1-1000 ng total HA) when the dots cut from the membrane were counted with liquid scintillation. Negligible interference from an excess of unincorporated precursor, chondroitin sulfate, and proteolytic tissue digest was observed. The assay was particularly convenient in measuring large numbers of samples with relatively low activity, such as testing the effects of different drug concentrations and analyzing chromatographic fractions.

Cetylpyridinium↗
PubMed1994

Purification of poly(ADP-ribose) glycohydrolase and detection of its isoforms by a zymogram following one- or two-dimensional electrophoresis.

Poly(ADP-ribosyl)ation metabolism, a post-translational modification, involves two nuclear enzymes. Poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) are responsible for the anabolism and catabolism of poly(ADP-ribose) polymer, respectively. PARG, despite being less abundant than PARP, is a crucial determinant of polymer metabolism which is known to be implicated in DNA repair and other cellular processes. Here, we describe modifications to improve the purification of PARG from calf thymus, in terms of both quantity and quality, which would allow biochemical and immunological studies. We also developed a zymogram to identify functional polypeptides exhibiting PARG activity. Purified and crude enzyme preparations from calf thymus were electrophoresed in two-dimensional gels. Samples were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing the polymer substrate in the form of automodified PARP after a nonequilibrium pH gradient electrophoresis. After renaturation of PARG in the gel, four isoforms of activity were clearly detected in the purified enzyme preparation. Even in the crude extract of the tissue, we could observe the major isoform of PARG. This technique will permit a better understanding of poly(ADP-ribose) catabolism and better characterization of PARG isoforms.

Animals↗
PubMed1994

Western blotting of formaldehyde-fixed neuropeptides as small as 400 daltons on gelatin-coated nitrocellulose paper.

A method is described for Western blotting of peptides as small as 400 daltons (Da). Peptides were separated by tricine-sodium dodecyl sulfate electrophoresis and electroblotted to gelatin-coated PH79 nitrocellulose paper (0.1 micron). The electroblotted peptides were fixed to the nitrocellulose paper for 5-10 min in 4% paraformaldehyde solution. Using anti-rabbit FMRF-amide (Phe-Met-Arg-Phe-NH2) as primary antibody, positive immunoreactivity was detected with an amplified alkaline phosphatase assay which was sensitive to at least 0.5 microgram FMRFamide/lane. When immunoreactivity was determined with 125I-protein A, it was possible to amplify and detect weak signals by increasing the autoradiography time. Therefore, using the 125I-protein A detection method, Western blot analysis of brain extracts from Lymnaea stagnalis (pond snail) and Poecilia reticulata (guppy) indicated the presence of four FMRFamide immunoreactive bands after a 7-day exposure to X-ray film. The most abundant peptide coelectrophoresed with the FMRFamide standard (M(r) 598.8 Da). In addition, this Western blotting procedure also detected APGWamide (Ala-Pro-Gly-Try-NH2; 428.5 Da) and [D-Ala2]-Leu-enkephalinamide (568.7 Da) with their respective specific antibodies.

Animals↗