Protein Molecular Weight Determination Research Articles

Kekerabatan merpati hias (Columba livia) dilihat dari profil protein darah

Objective: The purpose of this study was to predict the kinship of ornamental pigeons based on blood proteins. Methods: The research method used is exploratory descriptive with a laboratory observation-based approach. Research procedures include: Blood Sampling, Blood Protein Isolation, Purification, Measurement of concentration with a nanodrop spectrophotometer and Electrophoresis. Data analysis includes: Protein Band Profile and Determination of Protein Molecular Weight with RF formula and Relationship with MVSP version 3.22 program. Results : The protein profile in the plasma of ornamental pigeons is different from one type of pigeon to another. some of the protein appears in the ornamental pigeon species but does not appear in other breeds. Likewise with the target protein to be analyzed, some are expressed and some are not expressed in ornamental pigeons. Results based on the analysis of protein profiles and dendograms of the four specific proteins (hemoglobin, transferrin, posttransferrin, and immunoglobulin) contained in erythrocytes and blood plasma, it can be seen that there are two clusters. Cluster 1 is occupied by varieties of ornamental pigeons Moven and cluster 2 is occupied by varieties of ornamental pigeons Lahore, American fantail, Vienna, Indian Fantail, King, Capucine, Gondok and Modena. Conclusions: The results of the ornamental pigeon kinship prediction based on blood protein showed that each variety had different kinship relationships. King, Capucine, Gondok and Modena pigeons have the closest relationship, and Moven pigeons have the most distant relationship from the researched ornamental pigeons.

Profile of Kerandang Fish (Channa pleurophthalmus Blkr) Proteins from Central Kalimantan

Kerandang fish (Channa pleurophthalmus Blkr) as one of freshwater fish from the Channidae family which is very potential and abundant in Central Kalimantan. The aim of this research was to determine and obtain a picture of the protein profile Kerandang fish. The benefit of this research is that Kerandang fish can be utilized maximally as one of the nutritious foods to demand the nutritional needs of the people in Central Kalimantan. This research was conducted exploratively qualitative with the parameters of the proximate test, amino acids using HPLC and determination of protein molecular weight using SDS-PAGE. The results obtained for the proximate test were 76.48% water content, 16.35% protein content, 0.35% fat content and 6.82% carbohydrate content. The results for amino acids assay obtained 15 types of amino acids, 8 types of essential amino acids with the highest type of amino acid, Leucine 4.69% and 7 types of non-essential amino acids with the highest type of amino acid, glutamic acid 9.11%, and total acid amino 53.32%. The result of analysis of protein profile of Kerandang fish meat based on its molecular weight using SDS-PAGE there are 27 protein bands in the range of molecular weights 10.202 - 134.432 kDa.

Determination of Protein Molecular Weights on SDS-PAGE.

An apparent molecular weight (MW) of a protein can be determined from the migration distance of a protein complexed with a strong cationic detergent sodium dodecyl sulfate (SDS) separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This method was established around 1969 and has been utilized substantially even today because of its simplicity. During the following half a century, although it has been reported that many proteins show some deviation in MW when determined on SDS-PAGE especially when their peptide chains are posttranslationally modified, this versatile method is still being used very often in current biochemical works. In this protocol, a simple method to estimate MW by running SDS-PAGE of standard proteins is explained by an example in which proteins extracted from mouse retina were analyzed by two-dimensional isoelectric focusing (2-D IEF) SDS-PAGE followed by protein identification by peptide mass fingerprinting.

The Potential of Candida Biofilm Protein as Bioreceptor for Candidiasis Immunoassay

Abstract: Candidiasis or infection that is caused by Candida has become a new list of the therapeutical problems recently. The difficulties in diagnosing are the main cause of the unsatisfactory results from common therapies and diagnosis methods. This has urged researchers to find alternative ways in candidiasis diagnosis such as serology-based detection using antigen or antibody development. The aim of this study was to evaluate the potential of protein derived from Candida albicans biofilm as bioreceptor on candidiasis immunoassay through Dot Blot method. The research method used descriptive method with the following stages: (1) preparation of Candida albicans biofilm (2) extraction of Candida albicans protein through enzymatic and mechanical methods, (3) determination of protein molecular weight with SDS-PAGE (4) production of polyclonal anti- candida and (5) analysis of protein extract as bioreeceptor on dot blot. Profile of biofilm proteins on SDS-PAGE analysis were shown on molecular weight 27,42; 29,89; 38,10; 44,90; 48,75; 52,92; 55,14; 59,86; 70,56; 87,36; 102,54;115,05; 130,14;143,14;181,53 kD. There were differences in the intensity of dots in the control group (44070) and treatment groups (63170.5). It is noticeable that biofilm protein extract of C. albicans can be used for induction of anti-Candida polyclonal antibody production as the potential candidate of bioreceptor in candidiasis immunoassay. Keywords: SDS-PAGE, polyclonal antibody, immunoassay, dot blot, biofilm

Production of polyclonal antibodies against the BYMV isolate infecting gladiolus crop naturally and development of polyclonal antibodies based serological diagnostic system

In this study a bean yellow mosaic virus isolate infecting gladiolus crop with leaf mosaic and color breaking symptoms on flower was characterized based on mechanical transmission, symptomatology on hosts, and viron particle structure and capsid protein molecular weight determination. The virus isolate was successfully purified from the propagating host and purified viron particles were used for the production of polyclonal antibodies for the development of a cost effective and sensitive serological diagnostic system. The produced polyclonal antibodies would be useful for the assessment of BYMV infection in gladiolus crop and seed materials.

Performance on absolute scattering intensity calibration and protein molecular weight determination at BL16B1, a dedicated SAXS beamline at SSRF

The optical system and end-station of bending-magnet beamline BL16B1, dedicated to small-angle X-ray scattering (SAXS) at the Shanghai Synchrotron Radiation Facility, is described. Constructed in 2009 and upgraded in 2013, this beamline has been open to users since May 2009 and supports methodologies including SAXS, wide-angle X-ray scattering (WAXS), simultaneous SAXS/WAXS, grazing-incidence small-angle X-ray scattering (GISAXS) and anomalous small-angle X-ray scattering (ASAXS). Considering that an increasing necessity for absolute calibration of SAXS intensity has been recognized in in-depth investigations, SAXS intensity is re-stated according to the extent of data processing, and the absolute intensity is suggested to be a unified presentation of SAXS data in this article. Theory with a practical procedure for absolute intensity calibration is established based on BL16B1, using glass carbon and water as primary and secondary standards, respectively. The calibration procedure can be completed in minutes and shows good reliability under different conditions. An empirical line of scale factor estimation is also established for any specific SAXS setup at the beamline. Beamline performance on molecular weight (MW) determination is provided as a straightforward application and verification of the absolute intensity calibration. Results show good accuracy with a deviation of less than 10% compared with the known value, which is also the best attainable accuracy in recent studies using SAXS to measure protein MW. Fast MW measurement following the demonstrated method also enables an instant check or pre-diagnosis of the SAXS performance to improve the data acquisition.

Spent brewer's yeast extracts as a new component of functional food

The use of yeast extracts as a natural and valuable additive ingredient intended for the production of functional food and dietary supplements were demonstrated. The chemical composition, amino acid analysis, determination of protein molecular weights, antioxidant properties, and sensory evaluation were carried out for two yeast extracts. It was found that the tested extracts are characterised by high essential amino acid content, exceeding the levels of reference protein developed by the FAO/WHO, and high antioxidant activity. Sensory characteristics of tested extracts may favourably influence the quality of the proposed functional foods and dietary supplements. The obtained results indicate that the tested extracts can be utilised as a source of free amino acids and peptides in the design of functional foods and dietary supplements.

Quantitative determination of protein molecular weight with an acoustic sensor; significance of specific versus non-specific binding.

Surface acoustic wave sensors with integrated microfluidics for multi-sample sensing have been implemented in this work towards the quantitative correlation of the acoustic signal with the molecular weight of surface bound proteins investigating different interaction/binding conditions. The results are presented for: (i) four different biotinylated molecules (30 ≤ Mw ≤ 150 kDa) specifically binding to neutravidin; (ii) the same four non-biotinylated molecules, as well as neutravidin, adsorbing onto gold; and (iii) four cardiac marker proteins (86 ≤ Mw ≤ 540 kDa) specifically binding to their homologous antibodies. Surface plasmon resonance was employed as an independent optical mass sensor. A linear relationship was found to exist between the phase change of the acoustic signal and the molecular weight of the proteins in both cases of specific binding. In contrast, non-specific binding of proteins directly onto gold exhibited no such linear relationship. In all three cases phase change was correlated with the bound mass per area. The underlying mechanism behind the different behavior between specific and non-specific binding is discussed by taking into account the geometrical restrictions imposed by the size of the specific biorecognition molecule and the corresponding bound protein. Our results emphasize the quantitative nature of the phase of the acoustic signal in determining the Mw (in the case of specific binding) with a resolution of 15% and the mass of the bound proteins (in all cases), as well as the significance of the biorecognition molecules in deriving the molecular weight from acoustic or optical detectors.

Rapid Determination of Protein Molecular Weight by the Ferguson Method and Multiplexed Capillary Electrophoresis

A method based on the use of the Ferguson method and multiplexed sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) with UV detection was demonstrated for the rapid determination of molecular weights of proteins. The method employs a capillary array where different uncoated capillaries are filled with gel buffers containing different concentrations of poly(ethylene oxide) (PEO). All data required to construct Ferguson plots and universal calibration curves for the determination of the molecular weights of diverse types of proteins are generated simultaneously in an eight-capillary array within 20 min.

Isolation and Characterization of Soluble Proteins in Grapes, Grape Juice, and Wine

Improved sensitive techniques for the determination of soluble proteins in Gewürztraminer and White Riesling grapes, juice, and wine are reported. These techniques (<i>1</i>) minimize interference from phenolics and oxidase enzymes during extraction of proteins, (<i>2</i>) permit direct determination of proteins in juice and wine samples by electrophoretic techniques without prior pretreatment and concentration, (<i>3</i>) permit the detection of glycoprotein, and (<i>4</i>) permit the simultaneous determination of protein molecular weight (MW) and isoelectric point (pl) by two-dimensional isoelectric focusing-lithium dodecyl sulfate polyacrylamide gel electrophoresis (IF-LDS PAGE). Phenolic scavengers, Amberlite XAD-4 and polyvinylpolypyrrolidone (PVPP) and the enzyme inhibitors, phenylmethylsulfonyl fluoride (PMSF) and diethyldithiocarbamate (DECA), are used for protein isolation. One-dimensional 7.5% to 15% sucrose-stabilized linear gradient LDS PAGE with silver staining permits effective resolution of 3 µg of protein. The sensitivity for protein detection is increased 100-fold (detection at 10 ng level) by using silver staining instead of Coomassie blue. Changes in relative concentration of proteins on stained gels were determined by laser scanning densitometry. Two-dimensional IF-LDS PAGE, as well as protein blotting for glycoprotein detection, were used for further separation and characterization of the soluble proteins in wine. Grapes contain up to 41 protein fractions with MW in the range of 11 200 to 190 000; juices and wines from these grapes contain up to 25 protein fractions with MW in the range of 11 200 to 65 000. Each of these fractions were further separated into several fractions on the basis of their pl. A high proportion of wine protein fractions is of low pl (4.1 - 5.8) and low MW (20 000 - 30 000). Three protein fractions with MW of 12 600, 25 000, and 28 000 are identified as containing glycoproteins in Gewürztraminer wine. Only two fractions, with MW of 25 000 and 28 000, were detected as containing glycoproteins in White Riesling wine.

Peptide and protein molecular weight determination by electrophoresis using a high-molarity tris buffer system without urea

Various buffer systems were examined for their ability to resolve and provide molecular weight determinations of proteins and peptides over a wide size range using electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Sharp bands and high resolution were achieved in the 1300 to 100,000 molecular weight range using a modified Laemmli discontinuous buffer system with high concentrations of Tris in the resolving gel (0.75 m) and in the running buffer (0.05 m). Linear gradient gels (8 to 25% acrylamide) were tested with and without varying concentrations of urea and/or glycerol and/or sucrose. At this high molarity of Tris, the inclusion of urea, glycerol, or sucrose proved unnecessary for successful peptide electrophoresis. Gels run without these reagents showed superior resolution throughout the entire molecular weight range when run with Tris at 0.75 and 0.05 m, respectively, obviating the need for urea or other additives as used in other systems. A single gel is thus able to resolve an entire range from large proteins to small peptides.

3] Determination of protein molecular weight in complexes with detergent without knowledge of binding

This chapter is concerned with the use of sedimentation equilibrium, a procedure that is generally applicable to proteins in the molecular weight range 10,000 to 500,000 and can be used conveniently for protein-amphiphile complexes without interference from non-protein-containing detergent micelles. This chapter presents a summary of the theoretical treatment of sedimentation equilibrium in a centrifugal field for systems containing two or more components and discusses the effects of specific interactions between the protein and solute molecules. It then proceeds to a description of experimental procedures and present examples of the application of this technique to protein-amphiphile complexes. Partial specific volumes are tabulated for amino acid residues, carbohydrates, and a number of detergents and lipids. Atomic and group partial molar volumes that are used to calculate partial specific volumes for chemical compounds are also provided. The chapter's consideration is the determination of protein molecular weight in a homogeneous system by a rigorous thermodynamic method.

Effects of the estrous cycle and early pregnancy on bovine uterine, luteal, and follicular responses.

Uterine, luteal, and follicular responses associated with the estrous cycle and early pregnancy in cattle were examined. Dairy (n = 19) and beef (n 19) cattle were slaughtered either on Day 4, 8, 12, 14, 16, or 19 postestrus (estrus = Day o). Corpus Iuteum (CL) weight, specific PGF20 binding by the luteal particulate fraction (100,000 X g pellet), in vitro estradiol (E2) production by the two largest follicles, total recoverable uterine luminal protein (TP), total recoverable iinmunoreactive uterine luminal PGF (TPGF), and peripheral plasma steroids were evaluated. In a parallel study, beef cattle (n 22) were slaughtered either on Day 8, 12, 14, 16, or 19 of pregnancy for measurements of TP, TPGF, and plasma steroids. Uterine luminal proteins, from cyclic and pregnant cattle (Day 19), were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for determination of protein molecular weights and protein profile characterization. In cyclic cattle, CL regression was not completed by Day 19. Follicle E2 secretion varied among animals within day (,i = 4.0-24.0 ng E2 /follicle/3.5 h; P<0.O1), but not among days. Total PGF2a binding (fmole/CL) for Days 4, 8, 12, 14, 16, and 19 was 14.29, 145.79, 177.34, 111.82, 174.51, and 199.17 (P<O.ol). TPGF (ng) varied among cycle Days 4 (14.4), 8 (13.9), 12 (19.7), 14 (47.7), 16 (17.4), and 19 (111.0 ng: P<0.01). In contrast, TPGF (ng) from pregnant cattle was 481.6 and 1187.8 ng on Day 16 (n 5) and 19 (n = 6). TP (mg) in cyclic cattle varied (P<0.05) among Days 4 (7.34), 8 (7.03), 12 (4.14), 14(15.92), 16 (5.88), and 19 (11.35). Mean TP (mg) for pregnant cattle ranged from 2.73 to 12.09. Thirty-two protein categories were identified in cyclic cattle uterine flushings by SDS-PAGE (Mr range X i(T� = 18.7 to 292.0). Proteins appeared with greater frequency later in the cycle than earlier (Days 14, 16, 19: 60% vs Days 4, 8, 12: 45%). Composite SDS-PAGE profiles from Days 8-12 and Days 14-16 differed (P<0.10) suggesting luteal phase stimulation of protein secretion. Protein profiles (SDS-PAGE) from Day 19 of pregnancy differed from Day 19 of the estrous cycle, resembled those of midluteal phase, and revealed four protein constituents possibly unique to early pregnancy. In summary, uterine luminal PGF increased with luteal phase of the estrous cycle at a time when there appeared to be a stable population of specific PGF2a binding sites in the CL. Higher PGF in utero at Days 16 and 19 of pregnancy and changes in TP and SDS-PAGE protein profiles may reflect responsiveness of endometrium to changes in ovarian status (cyclic) and/or conceptus activity (pregnancy comparisons).

Aqueous N1-β-phenethylbiguanide hydrochloride as a solvent for protein molecular weight determination

Phenethylbiguanide ( N-(2-phenylethyl)imidodicarbonimidic diamide) hydrochloride was shown to be a potent denaturing agent, according to optical rotation measurements, which at a concentration of 1.17 m yielded the proper molecular weight of reduced-carboxymethylated hen's egg lysozyme as determined by osmotic pressure measurements. The conformation of the unfolded molecule was identical to that in 6 m guanidine hydrochloride, as was shown by intrinsic viscosity measurements. The relatively low density of the solvent, 1.06 g/ml, recommends it for use in sedimentation-equilibrium studies. It would be possible to use rotor speeds 15% lower than those required when the more commonly used solvent, 6 m guanidine hydrochloride (density ∼1.14 g/ml) is used.

Determination of protein molecular weights in denaturing solvents using glyceryl-CPG

Glyceryl-CPG is controlled-pore glass whose surface has been chemically modified to block its slight negative charge in aqueous solution. In contrast to normal controlled-pore glass, glyceryl-CPG can be used successfully for gel filtration of proteins in a variety of denaturing solvents, viz., 8 M urea, 6 M guanidine hydrochloride, and 0.1% sodium dodecyl sulphate. It has been found that glass with a mean pore diameter near 35 nm is suitable for the molecular weight range 17,000–100,000. Glyceryl-CPG with a smaller pore diameter is more satisfactory for molecular weights below 30,000. The stable pore size and bed dimensions of controlled-pore glass allows comparison of the conformation of a particular protein in different solvents to be made with the same column.

Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis

A method has been found which distinguishes between a size isomer family of proteins (e.g. bovine serum albumin polymers) and a charge isomer family of proteins (e.g. lactate dehydrogenase isoenzymes), utilizing disc gel electrophoresis. When the log of protein mobility relative to the dye front was plotted versus acrylamide gel concentration, size isomeric proteins gave a family of nonparallel lines extrapolating to a common point in the vicinity of 0% gel concentration; charge isomeric proteins gave a parallel family of lines. Proteins differing in both charge and size gave non-parallel lines intersecting at gel concentrations other than 0% gel concentration. The slope of such a plot is related to molecular weight. The molecular weight-slope relation was established utilizing 17 well-characterized proteins as standards. From this relation, it is possible to determine the molecular weight of a protein with an average precision of ±4%. This molecular weight method can be applied to a single protein in a mixture of proteins provided a specific detection test is available. This method should find uses in distinguishing between size and charge isomer families of proteins, for the rapid, easy, and accurate determination of protein molecular weights, and as a valuable aid in indicating the procedures to be used in enzyme purifications.

The determination of protein molecular weights of up to 225,000 by gel-filtration on a single column of Sephadex G-200 at 25° and 40°

The present paper describes the use of a cross-linked dextran-gel, Sephadex G-200, for the determination of protein molecular weights of up to 225,000. Evidence is provided which indicates that measurements beyond this are possible. A second system is described, employing a column temperature of 40° and a buffer of high ionic strength which is for use with proteins, such as gelatin, where aggregation effects must be eliminated.