scholarly journals Protein quantification and visualization via ultraviolet-dependent labeling with 2,2,2-trichloroethanol

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Anand Chopra ◽  
William G. Willmore ◽  
Kyle K. Biggar
Keyword(s):  
Light Exposure ◽  
Covalent Modification ◽  
Protein Quantification ◽  
Visible Range ◽  
Uv Absorbance ◽  
Polyacrylamide Gels ◽  
Fluorescent Emission ◽  
Tyrosine Residues ◽  
Sds Page ◽  
Low Volume

Abstract The incorporation of 2,2,2-trichloroethanol in polyacrylamide gels allows for fluorescent visualization of proteins following electrophoresis. Ultraviolet-light exposure, in the presence of this trichlorinated compound, results in a covalent modification of the tryptophan indole ring that shifts the fluorescent emission into the visible range. Based on this principle, we used 2,2,2-trichloroethanol to develop a microplate format protein quantification assay based on the fluorescent signal generated by modified proteins. We also demonstrated a specific fluorescent emission of 2,2,2-trichloroethanol-labeled protein at 450 nm, with a 310 nm excitation, resulting from modification of both tryptophan and tyrosine residues. Following optimization, this protein quantification assay displayed superior sensitivity when compared to UV absorbance at 280 nm (A280), and enabled quantification beyond the linear range permitted by the Bradford method. This 100 μL assay displayed a sensitivity of 10.5 μg in a range up to at least 200 μg. Furthermore, we extended the utility of this method through the development of a 20 μL low-volume assay, with a sensitivity of 8.7 μg tested up to 100 μg, which enabled visualization of proteins following SDS-PAGE. Collectively, these results demonstrate the utility of 2,2,2-trichloroethanol-based protein quantification and demonstrates the protein visualization in polyacrylamide gels based on 2,2,2-trichloroethanol-labeling pre-electrophoresis.

scholarly journals Heat and Pressure Treatments on Almond Protein Stability and Change in Immunoreactivity after Simulated Human Digestion

Nutrients ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 1679 ◽  
Author(s):  
Elisabetta De Angelis ◽  
Simona Bavaro ◽  
Graziana Forte ◽  
Rosa Pilolli ◽  
Linda Monaci
Keyword(s):  
Protein Stability ◽  
Total Protein ◽  
Protein Pattern ◽  
Protein Quantification ◽  
Protein Profiling ◽  
Enzyme Linked Immunosorbent Assay ◽  
Total Protein Content ◽  
Bioinformatic Tools ◽  
Sds Page ◽  
Allergenic Proteins

Almond is consumed worldwide and renowned as a valuable healthy food. Despite this, it is also a potent source of allergenic proteins that can trigger several mild to life-threatening immunoreactions. Food processing proved to alter biochemical characteristics of proteins, thus affecting the respective allergenicity. In this paper, we investigated the effect of autoclaving, preceded or not by a hydration step, on the biochemical and immunological properties of almond proteins. Any variation in the stability and immunoreactivity of almond proteins extracted from the treated materials were evaluated by total protein quantification, Enzyme Linked Immunosorbent Assay (ELISA), and protein profiling by electrophoresis-based separation (SDS-PAGE). The sole autoclaving applied was found to weakly affect almond protein stability, despite what was observed when hydration preceded autoclaving, which resulted in a loss of approximately 70% of total protein content compared to untreated samples, and a remarkable reduction of the final immunoreactivity. The final SDS-PAGE protein pattern recorded for hydrated and autoclaved almonds disclosed significant changes. In addition, the same samples were further submitted to human-simulated gastro-intestinal (GI) digestion to evaluate potential changes induced by these processing methods on allergen digestibility. Digestion products were identified by High Pressure Liquid Chromatography-High Resolution Tandem Mass Spectrometry (HPLC-HRMS/MS) analysis followed by software-based data mining, and complementary information was provided by analyzing the proteolytic fragments lower than 6 kDa in size. The autoclave-based treatment was found not to alter the allergen digestibility, whereas an increased susceptibility to proteolytic action of digestive enzymes was observed in almonds subjected to autoclaving of prehydrated almond kernels. Finally, the residual immunoreactivity of the GI-resistant peptides was in-silico investigated by bioinformatic tools. Results obtained confirm that by adopting both approaches, no epitopes associated with known allergens survived, thus demonstrating the potential effectiveness of these treatments to reduce almond allergenicity.

Urine protein quantification in stacking gel by SDS-PAGE

2018 ◽  
Vol 40 (4) ◽  
pp. 487-490
Author(s):  
Jia Jia ◽  
Jie Pan ◽  
Hongpan Xu ◽  
Sen Wang ◽  
Bing Bai
Keyword(s):  
Protein Quantification ◽  
Urine Protein ◽  
Sds Page

scholarly journals Tyrosine phosphorylation of P-selectin in intact platelets and in a disulphide-linked complex with immunoprecipitated pp60c-src

1994 ◽  
Vol 299 (3) ◽  
pp. 613-621 ◽  
Author(s):  
P W Modderman ◽  
A E G K von dem Borne ◽  
A Sonnenberg
Keyword(s):  
Tyrosine Phosphorylation ◽  
Cell Activation ◽  
Secretory Granules ◽  
Protein S ◽  
Kinase Assay ◽  
Intact Cells ◽  
Tyrosine Residues ◽  
Reducing Conditions ◽  
Sds Page

P-selectin is a 140 kDa membrane glycoprotein found in secretory granules of platelets and endothelial cells where it is rapidly translocated to the plasma membrane upon cell activation. It then functions as a receptor for various types of leucocytes. Metabolic labelling of resting platelets with 32Pi showed that P-selectin is primarily phosphorylated on serine residues, although some tyrosine phosphorylation was observed as well. However, tyrosine phosphorylation of P-selectin was greatly stimulated by treatment with the permeating phosphatase inhibitor, pervanadate. When P-selectin immunoprecipitates were incubated with [gamma-32P]ATP (in vitro kinase assay), a fraction of P-selectin was phosphorylated on its tyrosine residues by a co-precipitated kinase. P-selectin phosphorylated in vitro co-migrated with 140 kDa surface-labelled 125I-P-selectin during SDS/PAGE under reducing conditions. Under non-reducing conditions, however, phosphorylated P-selectin was disulphide-linked to unknown protein(s) in a 205 kDa complex. In vitro kinase assays of the most abundant platelet tyrosine kinase, pp60c-src, demonstrated the presence of similar 140 and 205 kDa phosphorylated proteins in SDS/PAGE under reducing and non-reducing conditions respectively. Extraction and reprecipitation studies with proteins phosphorylated in vitro indicated that P-selectin and pp60c-src form a 205 kDa 1:1 disulphide-linked complex. In the complex, pp60c-src autophosphorylation is inhibited and P-selectin is phosphorylated on tyrosine residues. As protein disulphides in the cytoplasm of intact cells are extremely rare, our results suggest that P-selectin and pp60c-src, which co-localize in platelet dense granules, may be non-covalently associated and spontaneously form disulphide bridges during lysis. In addition, the observed tyrosine phosphorylation of P-selectin in intact platelets suggests that its function might be regulated by phosphorylation by pp60c-src.

Fluorescent staining of glycoproteins in sodium dodecyl sulfate polyacrylamide gels by 4H-[1]-benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide

The Analyst ◽  
2014 ◽  
Vol 139 (11) ◽  
pp. 2764-2773 ◽  
Author(s):  
Zhongxin Zhu ◽  
Xuan Zhou ◽  
Yang Wang ◽  
Lisha Chi ◽  
Dandan Ruan ◽  
...  
Keyword(s):  
Carboxylic Acid ◽  
Sodium Dodecyl Sulfate ◽  
Staining Method ◽  
Sodium Dodecyl ◽  
Acid Hydrazide ◽  
Polyacrylamide Gels ◽  
Fluorescent Staining ◽  
Affinity Enrichment ◽  
Carboxylic Acid Hydrazide ◽  
Sds Page

A sensitive, specific, economic and MS compatible staining method for gel-separated glycoproteins by using BH was described and demonstrated by 1-D and 2-D SDS-PAGE, deglycosylation, glycoprotein affinity enrichment and LC-MS/MS, respectively.

scholarly journals Protein covalent modification of biologically active quinones

2004 ◽  
Vol 69 (11) ◽  
pp. 901-907 ◽  
Author(s):  
Dusan Sladic ◽  
Irena Novakovic ◽  
Zoran Vujcic ◽  
Tatjana Bozic ◽  
Natasa Bozic ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Chemical Modification ◽  
Covalent Modification ◽  
Biologically Active ◽  
Amino Groups ◽  
Sds Page ◽  
Protein Covalent

The avarone/avarol quinone/hydroquinone couple shows considerable antitumor activity. In this work, covalent modification of ?-lactoglobulin by avarone and its derivatives as well as by the synthetic steroidal quinone 2,5(10)-estradiene- 1,4,17-trione and its derivatives were studied. The techniques for studying chemical modification of ?-lactoglobulin by quinones were: UV/Vis spectrophotometry, SDS PAGE and isoelectrofocusing. SDS PAGE results suggest that polymerization of the protein occurs. It could be seen that the protein of 18 kD gives the bands of 20 kD, 36 kD, 40 kD, 45 kD, 64 kD and 128 kD depending on modification agent. The shift of the pI of the protein (5.4) upon modification toward lower values (from pI 5.0 to 5.3) indicated that lysine amino groups are the principal site of the reaction of ?-lactoglobulin with the quinones.

scholarly journals Multispectral estimation of retinal photoreceptoral inputs

2019 ◽  
Vol 11 (3) ◽  
pp. 60
Author(s):  
Salvador Bara ◽  
Maria Angeles Bonmati-Carrion ◽  
Juan Antonio Madrid ◽  
Maria Angeles Rol ◽  
Jaime Zamorano
Keyword(s):  
Ganglion Cells ◽  
Light Exposure ◽  
International Workshop ◽  
Optimal Estimation ◽  
Visible Range ◽  
Night Time ◽  
Software Applications ◽  
Age Trends ◽  
Key Aspects ◽  
20 Nm

Integrated multispectral devices provide convenient means for assessing the inputs to the five known photoreceptors present in the human retina. These photoreceptors drive relevant visual and non-visual pathways that control key aspects of human physiology. In this Letter we show that standard metrics of retinal photoreceptoral exposure can be quantitatively estimated, to within a 5%, by means of 12 multispectral channels, 20 nm wide (FWHM), distributed across the visible range. Full Text: PDF ReferencesD.M. Berson, F.A. Dunn, M. Takao, "Phototransduction by Retinal Ganglion Cells That Set the Circadian Clock", Science 295, 1070 (2002). CrossRef R.J. Lucas et al. "Measuring and using light in the melanopsin age", Trends in Neurosciences 37, 1 (2014). CrossRef Commission Internationale de l'Eclairage. TN 003:2015 Report on the First International Workshop on Circadian and Neurophysiological Photometry, 2013. CIE, 2015. DirectLink A. Sánchez de Miguel et al, "Evaluating Human Photoreceptoral Inputs from Night-Time Lights Using RGB Imaging Photometry", Journal of Imaging 5(4), 49 (2019). CrossRef M.S. Rea, M.G. Figueiro, A. Bierman, R. Hamner. "Modelling the spectral sensitivity of the human circadian system", Lighting Research and Technology 44, 386 (2012). CrossRef D. Cao, P.A. Barrionuevo, "Estimating photoreceptor excitations from spectral outputs of a personal light exposure measurement device", Chronobiology International 32(2), 270 (2015). CrossRef P.B. Liebelt, An Introduction to Optimal Estimation (Reading, MA, Addison-Wesley, 1967). CrossRef J. Escofet, S. Bará, "Reducing the circadian input from self-luminous devices using hardware filters and software applications", Lighting Research and Technology 49, 481 (2017). CrossRef

scholarly journals External morphological stages and protein variations along with the embryonic development of the longarm river prawn Macrobrachium tenellum (Smith, 1871)

2021 ◽  
Vol 49 (2) ◽  
pp. 202-211
Author(s):  
Marcelo U. García-Guerrero ◽  
Dulce M. Mateos-Guerrero ◽  
Juan J. Alpuche-Osorno ◽  
Rodolfo B. De los Santos-Romero
Keyword(s):  
Embryonic Development ◽  
Protein Concentration ◽  
Egg Size ◽  
Morphological Changes ◽  
Larval Rearing ◽  
Polyacrylamide Gels ◽  
Protein Extract ◽  
Molecular Weights ◽  
Sds Page ◽  
Page Electrophoresis

A good understanding of a given species' embryology is important to settle the larval rearing bases when juveniles are required for culture purposes or conservation programs. Changes in embryonic morphology, protein concentration, and protein type occurring in prawn eggs were analyzed in the present work. Berried females of Macrobrachium tenellum were collected in the Colotepec River, Oaxaca, Mexico. The eggs were taken from the ovigerous mass and embryonic stages classified by their color. Morphological changes in the embryos allowed identifying six embryonic stages based on color, egg size, and morphological features. Determinations of the protein extract were executed in SDS-PAGE (electrophoresis in polyacrylamide gels) and, subsequently, proteomic analyses were also performed. Protein bands along embryonic development and their molecular weights are presented and commented.

scholarly journals Investigating Toxin Diversity and Abundance in Snake Venom Proteomes

2022 ◽  
Vol 12 ◽  
Author(s):  
Theo Tasoulis ◽  
Tara L. Pukala ◽  
Geoffrey K. Isbister
Keyword(s):  
Snake Venom ◽  
Protein Identification ◽  
Protein Quantification ◽  
Clinical Effects ◽  
Protein Families ◽  
Crude Venom ◽  
Proteome Coverage ◽  
Current State ◽  
Sds Page ◽  
Viper Venoms

Understanding snake venom proteomes is becoming increasingly important to understand snake venom biology, evolution and especially clinical effects of venoms and approaches to antivenom development. To explore the current state of snake venom proteomics and transcriptomics we investigated venom proteomic methods, associations between methodological and biological variability and the diversity and abundance of protein families. We reviewed available studies on snake venom proteomes from September 2017 to April 2021. This included 81 studies characterising venom proteomes of 79 snake species, providing data on relative toxin abundance for 70 species and toxin diversity (number of different toxins) for 37 species. Methodologies utilised in these studies were summarised and compared. Several comparative studies showed that preliminary decomplexation of crude venom by chromatography leads to increased protein identification, as does the use of transcriptomics. Combining different methodological strategies in venomic approaches appears to maximize proteome coverage. 48% of studies used the RP-HPLC →1D SDS-PAGE →in-gel trypsin digestion → ESI -LC-MS/MS pathway. Protein quantification by MS1-based spectral intensity was used twice as commonly as MS2-based spectral counting (33–15 studies). Total toxin diversity was 25–225 toxins/species, with a median of 48. The relative mean abundance of the four dominant protein families was for elapids; 3FTx–52%, PLA2–27%, SVMP–2.8%, and SVSP–0.1%, and for vipers: 3FTx–0.5%, PLA2–24%, SVMP–27%, and SVSP–12%. Viper venoms were compositionally more complex than elapid venoms in terms of number of protein families making up most of the venom, in contrast, elapid venoms were made up of fewer, but more toxin diverse, protein families. No relationship was observed between relative toxin diversity and abundance. For equivalent comparisons to be made between studies, there is a need to clarify the differences between methodological approaches and for acceptance of a standardised protein classification, nomenclature and reporting procedure. Correctly measuring and comparing toxin diversity and abundance is essential for understanding biological, clinical and evolutionary implications of snake venom composition.

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