Browsing by Subject "SDS-PAGE"

Monoclonal antibodies (mAbs) are widely used in the treatment of several diseases such as cancer and autoimmune diseases. Due to their high prices and growing consumption, therapeutic mAbs have become potential targets of falsification. This generates a demand for quick and efficient analytical procedures for identifying and characterizing mAbs in a case of suspected falsification. The structure of therapeutic mAbs consists of human or murine IgG framework, where unique complementarity determining regions (CDRs) are engineered with different recombinant techniques. Given the complex nature of the mAbs, they must be identified using multiple complementary analytical methods. Ten full-sized therapeutic mAbs, Fab-fragment abciximab and CTLA4-Fc-fusion protein belatacept were studied in order to find analytical methods for efficient characterization and identification. All studied antibodies were characterized by their charge and molecular weight by isoelectric focusing (IEF) in polyacrylamide gels, native and reduced SDS-PAGE, and size exclusion chromatography (SEC). Six mAbs, abciximab and belatacept were digested with trypsin, and the cleaved peptides were further analysed by RPLC-MS. In addition, quantification methods including SEC peak area measurements and Bradford protein assay were performed for all antibodies. As expected, SDS-PAGE of non-reduced and reduced mAbs gave little distinction between the mAbs. Both methods were however shown to be useful in the identification of the mAb nature, as they confirmed the presence of heavy chains, light chains, and disulfide bonds. IEF showed potential in mAb identification, as clear, partly distinguished patterns of charge variants were obtained. However, some improvements to the pH gradient are needed to enable better separation and pI estimation of basic variants. Determination of molecular size with SEC was found to be difficult, as there seemed to be no consistency between the calculated molecular weights based on measured elution times, and the theoretical molecular weights. Nevertheless, SEC brings added value in mAb quantification and detection of protein aggregation and fragmentation. Finally, RPLC-MS analysis of tryptic peptides resulted in mAb identification, with the measured sequence coverage of 87-97 %. Identification process may be enhanced by focusing on the known CDR-peptides prior the constant frame peptides. Given the structural similarity of therapeutic mAbs, identification of an unknown mAb requires combination of multiple analytical methods. If available, the use of reference mAb product obtained from a reliable source is recommended, as the identification may be based on comparative analyses using simpler analytical steps, e.g. IEF, SDS-PAGE and SEC. If no reference product is available, identification of the mAb requires peptide mapping and determination of the CRD sequences by RPLC-MS analysis. Further research is needed to find a suitable set of analytical methods for identification of all therapeutic mAbs.

Recently, a new muscular disorder has been reported in chicken M. pectoralis major called Wooden Breast that might be linked to intensive growth selection. The purpose of this study was to find the influence of Wooden Breast condition on protein composition of the breast muscle with special emphasis on myofibrillar and sarcoplasmic protein fractions. For fulfilling the aims of the study, a total number of 34 pectoralis major muscles from both Wooden Breast and normal chickens (Ross 508, Aviagen Ltd, Scotland) were used to evaluate protein composition, protein profile of sarcoplasmic and myofibrillar proteins and their changes, by doing one-dimensional SDS-PAGE analysis. M. Pectoralis major defected by Wooden Breast demonstrated significant decline in sarcoplasmic (P?0.001) and myofibrillar (P?0.05) protein content, in comparison with normal breast muscles. Furthermore, gel electrophoresis indicated significant changes in the intensity of 18 sarcoplasmic protein bands and 7 myofibrillar protein bands. Majority of affected sarcoplasmic proteins were glycolytic enzymes. Moreover, impacted myofibrillar proteins play a role in muscle fibre integrity (e.g. desmin) or calcium signalling. Results of the present study also revealed desmin overexpression with coexistence of a desmin fragment in Wooden Breast which was confirmed by Western-blot technique. In conclusion, the findings of this study indicated defected chicken breast with Wooden Breast contained less protein and the composition differed compared to normal chicken breast muscle, which proves the influence of Wooden Breast condition on protein characteristics of chicken breast muscle. Further studies are needed to interpret the protein changes in the Wooden Breast and the possible role of these changes on metabolic and structural status of the muscle.

The literature review focused on the composition of the barley hordeins and the known extraction methods. The metal-catalysed oxidation of proteins and amino acids was also reviewed. The aim of the experimental part was to develop a simple selective extraction method for the B and C hordeins and to study how metal-catalysed oxidation affects these hordeins. Hulles barley cf. Jorma was selected as test material since milling of this cultivar was simple with a Brabender Junior mill. From the milled barley, water and salt-soluble material were removed and the rest was freeze-dried. The freeze-dried sample flour was studied by gel separation, precipitation and extraction with aqueous alcohols. The aqueous alcohol extracted the C hordeins completely although there was some B hordeins present. Two-dimensional electrophoresis showed that the isoelectric point of C hordeins was between the pH 5–7. Aqueous alcohols, extraction temperatures and pH were studied for hordein extraction. None of the studied methods improved the extraction of B and C hordeins. The hordein sample used in further experiments was extracted with 55% 2-propanol at 50 ºC. The metal-catalysed oxidation of the extracted hordeins was studied by using copper or iron as a catalyst and hydrogen peroxide or ascorbic acid as an oxidant. The reactions were analysed with SDS-PAGE and SE-HPLC. The results showed that the most effective reaction was with copper and hydrogen peroxide where the B and C hordeins were degraded efficiently after 24 hours of incubation. The results from SE-HPLC showed aggregated B hordeins in the extracted sample, which were partly degraded after two hours of incubation with hydrogen peroxide and copper. The results of this study indicated that the biggest groups of hordeins, the B and C hordeins, cannot be selectively separated with extraction. The barley hordeins efficiently degraded in the metal-catalysed oxidation with hydrogen peroxide and copper.

Quinoa is a South American crop plant and the abrasive pearling of its seeds produces abrasion waste as a side stream. The aim of this study was to examine the chemical and physical differences between two different side streams and extract some valuable components from two abrasive side streams. The side streams were obtained with two different pearling methods and are referred to in this study as fine and coarse side stream. Four different extraction media; water, 0.4 M sodium chloride, 70% (v/v) ethanol and pH 9.0 sodium hydroxide, were used solubilize protein and carbohydrates from the side streams. Electrophoresis (SDS-PAGE) was used to detect the protein composition of the side streams and ultrafiltration (UF) of aqueous extract together with UPLC of UF permeate were used to separate and detect mono- and disaccharides from the side streams. The coarse side stream had bigger particle size and higher density and it contained more protein and fat than the fine side stream which instead had higher ash and fibre content than the coarse side stream. Mainly albumins and globulins as well as some glutelins were detected from water, saline, and alkaline extracts of both side streams. The coarse side stream’s ultrafiltration permeate contained sucrose, fructose, and glucose - sucrose being most abundant and glucose least. The fine side stream’s permeate contained glucose and some fructose, but no sucrose. This study found quinoa pearling side streams to be a potential source for carbohydrates and protein, but the differences in the compositions of the side streams significantly affect their potential for wider use. Further studies are needed to investigate the protein yields and mono and disaccharide content of extracts with other extraction media. This study also raises further research topics, such as the effect of pre-treatment methods on the mono and disaccharide yields.

Palautusjuoma on harjoittelun jälkeen nautittava nestemäinen elintarvike. Sille ei ole virallista määritelmää, mutta sen tarkoituksena on sisältää tärkeitä ravintoaineita kuten aminohappoja ja hiilihydraatteja, jotka käynnistävät urheilusuorituksesta palautumisen. Palautusjuomat voidaan jakaa karkeasti juomajauheisiin ja valmiisiin juotaviin tuotteisiin. Kasvipohjaisen palautusjuoman pohjana voisi toimia kasviproteiiniuute. Tämän työn tarkoituksena oli tutkia erilaisten uutto-olosuhteiden vaikutusta kvinoaproteiinien uuttumiseen. Uute, jossa olisi korkein proteiinisaanto, voisi toimia teoreettisen palautusjuoman pohjana. Muuttujina käytettiin liuotin/siemen (w/w) -suhdetta (10:1 ja 20:1), liuotinta (pH 6,71 ja 9; NaCl-pitoisuus [0 mol/l ja 0,03 mol/l]) ja liuottimen lämpötilaa (25 °C ja 40 °C). Uuttoaika oli kaikissa näytteissä 30 min. Proteiinisaantoon tilastollisesti merkitsevästi vaikuttavat muuttujat (p < 0,05) olivat liuottimen pH ja lämpötila. Tämän tiedon perusteella uutetta valmistettiin lisää kahdeksan litraa, jonka muuttujien arvoina olivat pH 9, NaCl-pitoisuus 0 mol/l, 40 °C liuotin/jauho (w/w) -suhde 10:1. Uute ultrasuodatettiin 20 kDa:n cut off -arvon kalvolla ja suodatuksesta otettiin talteen retentaatti ja permeaatti. Retentaattia sumutuskuivattiin 984 g ja kuiva-ainesaanto oli 0,84 %, eli 8,2 g jauhetta. Proteiinisaanto oli 29,8 %. Sumutuskuivauksesta saadusta jauheesta määritettiin proteiinipitoisuus, kuiva-ainepitoisuus ja vaahtoavuusominaisuuksia. Pakkaskuivatusta retentaatista tehtiin viskositeetti ja saponiinipitoisuusmääritykset. Lisäksi pakkaskuivatusta retentaatista ja permeaatista tehtiin SDS-PAGE elektroforeesi, jotta nähtäisiin, miten proteiinit jakautuivat ultrasuodatuksessa permeaattiin ja retentaattiin. Sumutuskuivatun kvinoajauheen proteiinipitoisuus oli 39,1 %. SDS-PAGE -määrityksestä saadun proteiiniprofiilin perusteella proteiinit jäivät retentaattiin ja ultrasuodatus onnistui hyvin. Retentaatti sisälsi kvinoalle ominaisia proteiineja: globuliineja (55 kDa) ja chenopodiineja (31–33 kDa). Tämän tutkimuksen perusteella työssä käytetyistä hiotuista kvinoan siemenistä uuttui vain vähän proteiineja, mikä johti matalaan proteiinipitoisuuteen sekä uutteessa että jauheessa. Siemenet eivät soveltuneet matalan proteiinipitoisuuden vuoksi palautusjuoman raaka-aineeksi, mutta vaahdonmuodostusominaisuuksien perusteella kvinoa kuitenkin vaikuttaa lupaavalta raaka-aineelta elintarviketeollisuuteen, erityisesti leipomoteollisuuteen. Raaka-aine vaatii kuitenkin lisätutkimusta, ja tulevaisuudessa määrityksiä voisi mahdollisesti tehdä hiomattomista siemenistä, jolloin proteiinipitoisuus olisi lähtökohtaisesti korkeampi.