Techniques for analysis of proteins by SDS-polyacrylamide gel electrophoresis and Western blotting

The separation of mixtures of proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is a technique that is widely used—and, indeed, this technique underlies many of the assays and analyses that are described in this book. While SDS-PAGE is routine in many labs, a number of issues require consideration before embarking on it for the first time. We felt, therefore, that in the interest of completeness of this volume, a brief chapter describing the basics of SDS-PAGE would be helpful. Also included in this chapter are protocols for the staining of SDS-PAGE gels to visualize separated proteins, and for the electrotransfer of proteins to a membrane support (Western blotting) to enable immunoblotting, for example. This chapter is intended to complement the chapters in this book that require these techniques to be performed. Therefore, detailed examples of why and when these techniques could be used will not be discussed here.

Application of matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in determining arginine esterase from snake venom

Using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The homogeneities and molecular weights of three arginine esterases from snake venom, which possessing therapeutic use in myocardial infarction, were determined and compared, MALDI-TOF-MS is possessed of high accuracy, high sensitivity and rapidity. MALDI-TOF-MS and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) can provide complementary and confirmatory results information. MALDI-TOF-MS can be directly used as an important method for the purification of snake venom complexes successfully.

One-dimensional and two-dimensional polyacrylamide gel electrophoresis: a tool for protein characterisation in aquatic samples

Dissolved organic nitrogen (DON) represents the least understood part of the nitrogen cycle. Due to recent methodological developments, proteins now represent a potentially characterisable fraction of DON at the macromolecular level. We have applied polyacrylamide gel electrophoresis to characterise proteins in samples from a range of aquatic environments in the Everglades National Park, Florida, USA. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed that each sample has a complex and characteristic protein distribution. Some proteins appeared to be common to more than one site, and these might derive from dominant higher plant vegetation. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) provided better resolution; however, strong background hindered interpretation. Our results suggest that the two techniques can be used in parallel as a tool for protein characterisation: SDS-PAGE to provide a sample-specific fingerprint and 2D-PAGE to focus on the characterisation of individual protein molecules.

Congruence between starch gel and polyacrylamide gel electrophoresis in detecting allozyme variation in pulmonate land slugs

The predominantly selfing slug species Arion (Carinarion) fasciatus, A. (C.) silvaticus and A. (C.) circumscriptus are native in Europe and have been introduced into North America, where each species consists of a single, homozygous multilocus genotype (strain), as defined by starch gel electrophoresis (SGE) of allozymes. In Europe, the “one strain per species” hypothesis does not hold since polyacrylamide gel electrophoresis (PAGE) of allozymes uncovered 46 strains divided over the three species. However, electrophoretic techniques may differ in their ability to detect allozyme variation. Therefore, several Carinarion populations from both continents were screened by applying the two techniques simultaneously on the same individual slugs and enzyme loci. SGE and PAGE yielded exactly the same results, so that the different degree of variation in North American and European populations cannot be attributed to differences in resolving power between SGE and PAGE. We found four A. (C.) silvaticus strains in North America indicating that in this region the “one strain per species” hypothesis also cannot be maintained. Hence, the discrepancies between previous electrophoretic studies on Carinarion are most likely due to sampling artefacts and possible founder effects.

Molecular weight estimation of esterase isoenzymes in closely related Drosophila Species (Diptera: Drosophilidae) in non-denaturing polyacrylamide gel electrophoresis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

A rapid and efficient two-step gel electrophoresis method for the purification of major rye grass pollen allergens

Purified proteins are mandatory for molecular, immunological and cellular studies. However, purification of proteins from complex mixtures requires specialised chromatography methods (i.e., gel filtration, ion exchange, etc.) using fast protein liquid chromatography (FPLC) or high-performance liquid chromatography (HPLC) systems. Such systems are expensive and certain proteins require two or more different steps for sufficient purity and generally result in low recovery. The aim of this study was to develop a rapid, inexpensive and efficient gel-electrophoresis-based protein purification method using basic and readily available laboratory equipment. We have used crude rye grass pollen extract to purify the major allergens Lol p 1 and Lol p 5 as the model protein candidates. Total proteins were resolved on large primary gel and Coomassie Brilliant Blue (CBB)-stained Lol p 1/5 allergens were excised and purified on a secondary "mini"-gel. Purified proteins were extracted from unstained separating gels and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses. Silver-stained SDS-PAGE gels resolved pure proteins (i.e., 875 μg of Lol p 1 recovered from a 8 mg crude starting material) while immunoblot analysis confirmed immunological reactivity of the purified proteins. Such a purification method is rapid, inexpensive, and efficient in generating proteins of sufficient purity for use in monoclonal antibody (mAb) production, protein sequencing and general molecular, immunological, and cellular studies.

Acute phase protein profile in agoutis (Dasyprocta azarae; Lichtenstein, 1823) in captivity, determined by sodium dodecyl sulfate polyacrilamyde gel electrophoresis

The acute phase response refers to a nonspecific and complex systemic reaction of the organism that occurs shortly after any tissue injury. The acute phase response is considered a part of the innate host defense system, which is responsible for the survival of the host during the critical early stages of attack, and in evolutionary terms, it precedes the acquired immune response. The purpose of this study was to determine serum protein concentrations, including the acute phase protein profile in agoutis (Dasyprocta azarae) in captivity, by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis. Blood samples from 11 adult healthy animals (nine females and two males) were obtained. The serum proteinogram had 21 proteins with molecular weights ranging from 15 to 240 kD. The acute phase proteins identified were: ceruloplasmin, transferrin, albumin, haptoglobin, α-1-acid glycoprotein, and hemoglobin. IgA, IgG heavy and light chains, and nonnominal identified proteins of 240, 210, 140, 98, 78, 48, 35, 31, 23, and 15 kD were also identified. The determination of the acute phase protein concentrations is a useful method for the early detection of subclinical disease or changes in the healthy animal, with predictive information on the development of disease in the future. It is possible to standardize the reference values of the serum protein profile of agoutis, which can be used for diagnosis and prognosis, treatment and clinical follow-up of nutritional disorders, and immune-mediated inflammatory diseases that may affect these animals. © 2012 Springer-Verlag London Limited.

Catalytic properties of 26 S and 20 S proteasomes and radiolabeling of MB1, LMP7, and C7 subunits associated with trypsin-like and chymotrypsin-like activities*

20 and 26 S proteasomes were isolated from rat liver. The procedure developed for the 26 S proteasome resulted in greatly improved yields compared with previously published methods. A comparison of the kinetic properties of 20 and 26 S proteasomes showed significant differences in the kinetic characteristics with certain substrates and differences in the effects of a protein substrate on peptidase activity. Observed differences in the kinetics of peptidylglutamyl peptide hydrolase activity suggest that the 26 S complex cannot undergo the conformational changes of 20 S proteasomes at high concentrations of the substrate benzyloxycarbonyl (Z) -Leu-Leu-Glu-b-naphthylamide. Various inhibitors that differentially affect the trypsin-like and chymotrypsin-like activities have been identified. Ala-Ala-Phe-chloromethyl (CH2Cl) inhibits chymotrypsin-like activity assayed with succinyl (Suc) -Leu-Leu-Val-Tyr-AMC, but surprisingly not hydrolysis of Ala-Ala-Phe-7-amido-4-methylcoumarin (AMC). Tyr-Gly-Arg-CH2Cl inhibits Suc-Leu-Leu-Val-Tyr-AMC hydrolysis as well as trypsinlike activity measured with t-butoxycarbonyl (Boc) -Leu-Ser-Thr-Arg-AMC, while Z-Phe-Gly-Tyr-diazomethyl (CHN2) was found to inhibit only the two chymotrypsin- like activities. Radiolabeled forms of peptidyl chloromethane and peptidyl diazomethane inhibitors, [3H]acetyl-Ala-Ala-Phe-CH2Cl, [3H]acetyland radioiodinated Tyr-Gly-Arg-CH2Cl, and Z-Phe-Gly- Tyr-(125I-CHN2), have been used to identify catalytic components associated with each of the three peptidase activities. In each case, incorporation of the label could be blocked by prior treatment of the proteasomes with known active site-directed inhibitors, calpain inhibitor 1 or 3,4-dichloroisocoumarin. Subunits of labeled proteasomes were separated either by reverse phase-HPLC and SDS-polyacrylamide gel electrophoresis or by twodimensional polyacrylamide gel electrophoresis followed by autoradiography/fluorography and immunoblotting with subunit-specific antibodies. In each case, label was found to be incorporated into subunits C7, MB1, and LMP7 but in different relative amounts depending on the inhibitor used, consistent with the observed effects on the different peptidase activities. The results strongly suggest a relationship between trypsin-like activity and chymotrypsin-like activity. They also help to relate the different subunits of the complex to the assayed multicatalytic endopeptidase activities

Heme biosynthesis by the malarial parasite - Import of delta-aminolevulinate dehydrase from the host red cell

The mouse and human malarial parasites, Plasmodium berghei and Plasmodium falciparum, respectively, synthesize heme de novo following the standard pathway observed in animals despite the availability of large amounts of heme, derived from red cell hemoglobin, which is stored as hemozoin pigment, The enzymes, delta-aminolevulinate dehydrase (ALAD), coproporphyrinogen oxidase, and ferrochelatase are present at strikingly high levels in the P, berghei infected mouse red cell in vivo, The isolated parasite has low levels of ALAD and the data clearly indicate it to be of red cell origin. The purified enzyme preparations from the uninfected red cell and the parasite are identical in kinetic properties, subunit molecular weight, cross-reaction with antibodies to the human enzyme, and N-terminal amino acid sequence. Immunogold electron microscopy of the infected culture indicates that the enzyme is present inside the parasite and, therefore, is not a contaminant, The parasite derives functional ALAD from the host and the enzyme binds specifically to isolated parasite membrane in vitro, suggestive of the involvement of a receptor in its translocation into the parasite, While, ALAD, coproporphyrinogen oxidase, and ferrochelatase from the parasite and the uninfected red cell supernatant have identical subunit molecular weights on SDS-polyacrylamide gel electrophoresis and show immunological cross-reaction with antibodies to the human enzymes, as revealed by Western analysis, the first enzyme of the pathway, namely, delta-aminolevulinate synthase (ALAS) in the parasite, unlike that of the red cell host, does not cross-react with antibodies to the human enzyme, However, ALAS enzyme activity in the parasite is higher than that of the infected red cell supernatant. We therefore conclude that the parasite, while making its own ALAS, imports ALAD and perhaps most of the other enzymes of the pathway from the host to synthesize heme de novo, and this would enable it to segregate this heme from the heme derived from red cell hemoglobin degradation, ALAS of the parasite and the receptor(s) involved in the translocation of the host enzymes into the parasite would be unique drug targets.

Ribosomal phosphoproteins in Microsporum canis

Ribosomal phosphoproteins of Microsporum canis labelled in vivo were characterised by two-dimensional and SDS polyacrylamide gel electrophoresis. A small subunit protein, S6, was the only phosphoprotein identified in 40S and 80S in basic-acidic two-dimensional gels. Three different forms of phosphorylated S6 were also observed in 40S subunit. On SDS gels five phosphoproteins were identified in 80S; of these three were present in 40S and two in 60S. S6 was the only basic phosphoprotein, while the other four were acidic.

Characterization of the DNA-binding domain of beta protein, a component of phage lambda Red-pathway by UV catalyzed cross-linking

beta protein, a key component of Red-pathway of phage lambda is necessary for its growth and general genetic recombination in recombination-deficient mutants of Escherichia coli. To facilitate studies on structure-function relationships, we overexpressed beta protein and purified it to homogeneity. A chemical cross-linking reagent, glutaraldehyde, was used to stabilize the physical association of beta protein in solution. A 67-kDa band, corresponding to homodimer, was identified after separation by SDS-polyacrylamide gel electrophoresis. Stoichiometric measurements indicated a site-size of 1 monomer of beta protein/5 nucleotide residues. Electrophoretic gel mobility shift assays suggested that beta protein formed stable nucleoprotein complexes with 36-mer, but not with 27- or 17-mer DNA. Interestingly, the interaction of beta protein with DNA and the stability of nucleoprotein complexes was dependent on the presence of MgCl2, and the binding was abolished by 250 mM NaCl. The K-d of beta protein binding to 36-mer DNA was on the order of 1.8 x 10(-6) M. Photochemical cross-linking of native beta protein or its fragments, generated by chymotrypsin, to 36-mer DNA was performed to identify its DNA-binding domain. Characterization of the cross-linked peptide disclosed that amino acids required for DNA-binding specificity resided within a 20-kDa peptide at the N-terminal end. These findings provide a basis for further understanding oi the structure and function of beta protein.

Cloning and characterization of a blood coagulation factor IX-binding protein from the venom of Trimeresurus stejnegeri

A blood coagulation factor IX-binding protein (TSV-FIX-BP) was isolated from the snake venom of Trimeresurus stejnegeri. On SDS-polyacrylamide gel electrophoresis, TSV-FIX-BP showed a single band with an apparent molecular weight of 23,000 under non-reducing conditions. and two distinct bands with apparent molecular weights of 14,800 and 14,000 under reducing conditions. cDNA clones containing the coding sequences of TSV-FIX-BP were isolated and sequenced to determine the structure of the precusors of TSV-FIX-BP subunits. The deduced amino acid sequences of two subunits of TSV-FIX-BP were confirmed by N-terminal protein sequencing and trypsin-digested peptide mass fingerprinting. TSV-FIX-BP was a nonenzymatic C-type lectin-like anti-coagulant. The anti-coagulant activity of TSV-FIX-BP was mainly caused by its dose dependent interaction with blood coagulation factor IX but not with blood coagulation factor X. (C) 2003 Elsevier Science Ltd. All rights reserved.

Molecular cloning and characterization of a platelet glycoprotein Ib-binding protein from the venom of Trimeresurus stejnegeri

A platelet glycoprotein Ib-binding protein, termed TSV-GPIb-BP, was isolated from the venom of Trimeresurus stejnegeri. On SDS-polyacrylamide gel electrophoresis, TSV-GPIb-BP showed a single band with an apparent molecular weight of 28,000 and two distinct bands with apparent molecular weights of 16,000 and 15,000 under non-reducing and reducing conditions, respectively. cDNA clones containing the coding sequences for both TSV-GPIb-BP subunits were isolated and sequenced. The deduced amino acid sequences of TSV-GPIb-BP subunits were confirmed by N-terminal protein sequencing and trypsin-digested peptide mass fingerprinting. Interestingly, the a subunit of TSV-GPIb-BP is identical to that of alboaggregin-B, and the sequence identity of their beta subunits is 94.3%. TSV-GPIb-BP inhibited ristocetin-induced human platelet agglutination in platelet-rich plasma under lower dosages (<5 mug/ml). On the other hand, it directly aggregated washed human platelets in the absence of additional Ca2+ or any other cofactors under higher dosages (>5 mug/ml). This platelet aggregation activity was dose-dependently inhibited by specific GPIbalpha antibodies, but not by those antibodies against platelet GPIa, GPIIa, GPIIb and GPIIIa. (C) 2003 Elsevier Science Ltd. All rights reserved.

Characterization and molecular cloning of dabocetin, a potent antiplatelet C-type lectin-like protein from Daboia russellii siamensis venom

A novel C-type lectin-like protein, dabocetin, was purified from Daboia russellii siamensis venom. On SDS-polyacrylamide gel electrophoresis, it showed a single band with an apparent molecular weight of 28 kDa and two distinct bands with the apparent mole