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 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.

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.

A Strong Correlation Between Spot Urine Protein To Creatinine Ratio and 24-Hour Urine Protein Quantification For Proteinuria Assessment In Patients With Plasma Cell Dyscrasia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3155-3155
Author(s):  
Apuri Susmitha ◽  
Taiga Nishihori ◽  
Karen Lin ◽  
Rachid Baz ◽  
Binglin Yue ◽  
...  
Keyword(s):  
Correlation Coefficient ◽  
Plasma Cell ◽  
Protein Quantification ◽  
Urine Samples ◽  
Urine Collection ◽  
Spearman Correlation ◽  
Urine Protein ◽  
Monoclonal Protein ◽  
Spot Urine ◽  
M Spike

Abstract Background Twenty four-hour urine collection remains one of the most crucial tools for the diagnosis and monitoring of proteinuria and quantification of urinary monoclonal protein in patients with plasma cell dyscrasias (PCD) even though it is cumbersome. Nephrology guidelines recommend replacement of 24-hour urine collection with a spot urine protein/creatinine (Pr/Cr) ratio for the measurement of proteinuria. However, only limited data exist regarding accuracy of spot urine Pr/Cr ratio in patients with PCD and utility of this measure to estimate urinary paraprotein remains uncertain. We conducted a prospective study evaluating the role of spot urine Pr/Cr ratio in patients with PCD. Methods From 04/2012 to 05/2013, a total of 120 PCD patients were prospectively enrolled at Moffitt Cancer Center. Oliguric patients or those requiring dialysis were ineligible. Spot urine was collected on the same day as 24-hour urine collection. Spot urine Pr/Cr ratios were compared to 24-hour urine collections with regard to the amount of (1) total protein and (2) monoclonal protein (M-spike). Results Eighty four out of 120 patients (70%) were evaluable (17 did not provide spot urine samples; no Pr/Cr ratios available in 11; protein below the level of detection in 7; and one without 24-hour urine collection). Evaluable patients had a median age of 68 (range, 36 - 90) years, 63% were male, and 85% were Caucasian. Primary diagnoses were myeloma (n=74; 88%), amyloidosis (n=4), multiple plasmacytomas (n=2), solitary plasmactyoma (n=1) and MGUS (n=3). Prior therapies included chemotherapy in 95% and autologous transplant in 45%. Comorbidities included hypertension (48%), chronic kidney disease (30%), diabetes (15%), coronary artery disease (8%), atrial fibrillation (7%) and congestive heart failure (2%). The median serum creatinine was 0.9 mg/dL (range, 0.5 - 5.1). The median spot urine Pr/Cr ratio was 137 mg/g creatinine (range, 26 - 21,447). The median 24-hour urine protein was 120 mg/24h (range, 27 - 15,092), and the median urine M-spike was 1.2 mg (range, 0 - 8,099). More than half of spot urine samples were collected in the morning (59%). There were strong correlations between (1) spot urine Pr/Cr ratio and 24-hour total urine protein (Spearman correlation coefficient=0.91, p < 0.0001), and (2) spot urine Pr/Cr ratio and 24-hour urine M-spike (Spearman correlation coefficient=0.91, p < 0.0001). The timing of spot urine sample collection had no significant effects on the correlations (p = 0.46 and 0.95 by Wilcoxon rank-sum test). Conclusions Spot urine Pr/Cr ratio strongly correlates with degrees of proteinuria measured in 24-hour urine collection and may also predict the quantity of urine M-spike in non-oliguric PCD population. Spot urine Pr/Cr ratio is a potentially useful marker as a screening tool or an alternative semi-quantitative measure for rapid estimation of proteinuria which may be used for longitudinal patient follow-up in lieu of cumbersome repeated 24-hour collections. Disclosures: No relevant conflicts of interest to declare.

scholarly journals AB0299 QUANTITATIVE EVALUATION OF PROTEINURIA WITH URINALYSIS TEST AND COMPARING ITS CORRELATION WITH RANDOM SPOT URINE PROTEIN-CREATININE RATIO AND 24 HOUR URINE PROTEIN IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS –A SINGLE CENTRE EXPERIENCE IN MALAYSIA

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 1176.1-1176
Author(s):  
C. R. Ng ◽  
Y. L. Loh
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Lupus Nephritis ◽  
Lupus Erythematosus ◽  
Standard Test ◽  
Protein Quantification ◽  
Nephrol Dial Transplant ◽  
Creatinine Ratio ◽  
Systemic Lupus ◽  
Urine Protein ◽  
Spot Urine

Background:Lupus nephritis is an important concern among Systemic Lupus Erythematosus (SLE) patients in Asia and its mortality rate was reported to be 6 times higher compared to the general population [1]. Without prompt treatment it can lead to end stage renal failure and affect quality of life. 24 hour urine protein collection has long been used as the gold standard test to assess proteinuria. However due to its cumbersome process random spot urine protein-creatinine ratio is used as an alternative to replace the former in some centres before subjecting patients to renal biopsy. In a study done by Matar HE et al in 2012, he showed that there was a significant correlation between 24 hour urine protein and urine protein creatinine ratio in his 95 subjects [2].Urinalysis is a semi-quantitative screening tool for early detection of potential kidney disorders. A survey done by Siedner MJ et al on practice preferences among American Rheumatologists in 2005 reported that 64.6% of them preferred to use urinalysis as the primary tool to screen for proteinuria [3].Objectives:To assess the correlation of urinalysis test with random spot urine protein-creatinine ratio PCR) compared with 24 hour urine protein.Methods:This was a retrospective study. The electronic medical records of all SLE patients seen in the rheumatology clinic of Hospital Sultan Ismail from 1/1/2017 to 31/12/2020 were reviewed. Patients who had urinalysis, urine protein creatinine ratio and 24 hour urine protein tests done were identified. Data on demography, urinalysis, random spot urine protein creatinine ratio and 24 hour urine protein were obtained and analysed.Results:There were a total of 131 patients and 124 were females. The majority were Malays (75/131) followed by the Chinese (45/131),Indians (9/141) and others (2/131). The mean age group for the studied subjects was 34 (13-67). The urinalysis test showed that 34 of them had negative results, 37 of them had urine protein of 1 +, 18 of them had urine protein of 2+ followed by 23 patients with urine protein of 3 + and the rest had urine protein of 4+. The correlation between urinalysis and 24 hour urine protein was strong (r = 0.702), whereas the correlation between urinalysis and urine PCR ratio was stronger (r = 0.797).Conclusion:We conclude that urinalysis correlates well with both random spot urine protein creatinine ratio and 24 hour urine protein and the correlation is stronger with urine PCR.References:[1]Yap DY, Tang CS, Ma MK, Lam MF, Chan TM. Survival analysis and causes of mortality in patients with lupus nephritis. Nephrol Dial Transplant. 2012;27:3248–3254. [PubMed][2]Matar HE, Peterson P, Sangle S, D’Cruz DP. Correlation of 24-hour urinary protein quantification with spot urine protein: creatinine ratio in lupus nephritis. Lupus 2012 Jul;21(8): 836-9. doi:10.1177/0961203312437438. Epub 2012 Feb 13.[3]Siedner MJ, Christopher-Stine L, Astor BC, Gelber AC, Fine DM. Screening for proteinuria in patients with lupus: a survey of practice preferences among American rheumatologists. J Rheumatol. 2007;34:973–977. [PubMed].Disclosure of Interests:None declared

scholarly journals Noninvasive sampling method for urinalysis and urine protein profile in captive giraffes

2020 ◽  
Vol 33 (1) ◽  
pp. 25-34
Author(s):  
Sabrina Fasoli ◽  
Enea Ferlizza ◽  
Giulia Andreani ◽  
Camillo Sandri ◽  
Francesco Dondi ◽  
...  
Keyword(s):  
Protein Profile ◽  
Urine Samples ◽  
Urine Specific Gravity ◽  
Urine Protein ◽  
Noninvasive Methods ◽  
Reliable Technique ◽  
Urine Creatinine ◽  
Sds Page ◽  
In Captivity ◽  
Animal Handling

Urinalysis could be helpful to investigate the health status of giraffes held in captivity using noninvasive methods to avoid animal handling or anesthesia. We collected 52 voided urine samples from 20 giraffes of different ages, sexes, and subspecies from the ground. To evaluate potential interference by soil contaminants, a pilot study was performed using 20 urine samples obtained from 10 cows. All bovine and 29 giraffe samples were subjected to routine urinalysis including urine specific gravity (USG). All samples were analyzed for urine total protein (uTP), urine creatinine (uCrea) concentration, and urine protein-to-urine creatinine ratio (UPC). Urinary proteins were separated by SDS-PAGE electrophoresis. No significant differences were determined between free-catch and urine sampled from the ground in cows. Giraffe urine was pale-yellow, with alkaline pH (>8.0) and a mean USG of 1.035 ± 0.013. The uTP, uCrea, and UPC expressed as median (range) were 0.20 (0.08–0.47) g/L, 2.36 (0.62–5.2) g/L, and 0.08 (0.05–0.15), respectively. SDS-PAGE allowed the separation of protein bands with different molecular masses, including putative uromodulin at 90 kD, putative albumin at 64 kD, and putative immunoglobulin heavy and light chains at 49 kD and 25 kD, respectively. Urine collection from the ground appears to be a reliable technique for urinalysis and urine electrophoresis in giraffes.

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.

Evaluation of spot urine protein to creatinine ratio and 24-hour urine protein quantification for proteinuria in patients with plasma cell dyscrasia.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e19516-e19516
Author(s):  
Susmitha Apuri ◽  
Taiga Nishihori ◽  
Karen Yin Lin ◽  
Rachid C. Baz ◽  
Jongphil Kim ◽  
...  
Keyword(s):  
Correlation Coefficient ◽  
Plasma Cell ◽  
Protein Quantification ◽  
Plasma Cell Dyscrasia ◽  
Urine Collection ◽  
Spearman Correlation ◽  
Sample Collection ◽  
Urine Protein ◽  
Spot Urine ◽  
M Spike

e19516 Background: Though cumbersome, a 24-hour urine collection remains crucial for diagnosis and monitoring in plasma cell dyscrasias (PCD). Nephrology guidelines recommend replacement of 24-hour urine collection with spot urine protein/creatinine (Pr/Cr) ratio for proteinuria. Only limited data exist on accuracy of spot urine Pr/Cr ratio in PCD. Methods: From 04/2012 to 01/2013, a total of 67 PCD patients were prospectively enrolled. Oliguria or dialysis were considered ineligible. Spot urine was collected on the day of 24-hour urine collection. Spot urine Pr/Cr ratios were compared to 24-hour urine on (1) total protein and (2) monoclonal protein (M-spike). Results: Fifty-two of 67 patients were evaluable (missing samples in 9; no Pr/Cr ratios in 4; protein below the level of detection in 2). A median age was 66 (range, 36 - 82), 62% were male, and 81% were Caucasian. Primary diagnoses were myeloma (n=47), amyloidosis (n=2), plasmacytoma (n=2), and MGUS (n=1). Comorbidities included hypertension (58%), chronic kidney disease (27%), diabetes (19%), and congestive heart failure (2%). A median serum creatinine was 0.9 mg/dL (range, 0.5 - 5.1). A median spot urine Pr/Cr ratio was 140 mg/g creatinine (range, 32 - 21,447), a median 24-hour urine protein was 130 mg (range, 31 - 15,092: n=49), and a median urine M-spike was 1.9 mg (range, 0 - 8,099). Most spot urine samples were collected in the morning (65%). There were strong correlations between (1) spot urine Pr/Cr ratio and 24-hour total urine protein (Spearman correlation coefficient=0.91, p < 0.0001), and (2) spot urine Pr/Cr ratio and 24-hour urine M-spike (Spearman correlation coefficient=0.73, p < 0.0001). The timing of spot urine sample collection had no significant effect (p = 0.274 by Wilcoxon rank-sum test). Conclusions: Spot urine Pr/Cr ratio strongly correlates with proteinuria measured in 24-hour urine collection and may predict the quantity of urine M-spike in non-oliguric PCD population. Spot urine Pr/Cr ratio may be useful as a monitoring tool for estimation of proteinuria. The study is ongoing and updated result will be provided.

scholarly journals Investigation of Heat and Pressure Treatments on Almond Protein Stability and Immunoreactivity after Simulated Human Digestion

2018 ◽  
Author(s):  
Elisabetta De Angelis ◽  
Simona L. Bavaro ◽  
Graziana Forte ◽  
Rosa Pilolli ◽  
Linda Monaci
Keyword(s):  
Total Protein ◽  
Protein Pattern ◽  
Protein Quantification ◽  
Protein Profiling ◽  
Total Protein Content ◽  
Bioinformatic Tools ◽  
Sds Page ◽  
Allergenic Proteins ◽  
The Stability

Almond is worldwide consumed and renowned as a valuable healthy food. In spite of this, it is also a potent source of allergenic proteins able to 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, ELISA assay and protein profiling by electrophoresis-based separation (SDS-PAGE). The autoclaving alone was found to weakly affect almond proteins stability, despite what observed for the combination of hydration and autoclaving, which resulted in a loss of approximately 70% of total protein content compared to untreated sample, and in a final negligible immunoreactivity, as well. The final SDS-PAGE protein pattern recorded for almonds hydrated and autoclaved disclosed significant changes. In addition, the same samples were further submitted to in vitro simulated gastro-duodenal (GI) digestion to evaluate potential changes induced by these processing on allergens digestibility. Digestion products were identified by HPLC-HRMS/MS analysis followed by software-based data mining, and complementary information were provided by analyzing the proteolytic fragments lower that 6 kDa in size. The autoclave based treatment was found not to alter the allergens digestibility, whereas an increased susceptibility to proteolytic action of digestive enzymes was observed in almonds subjected to the combination of prehydration and autoclaving. Finally, the residual immunoreactivity of the GI resistant peptides was investigated in-silico by bioinformatic tools, confirming that by following both approaches, no epitopes survived the almond digestion, thus demonstrating the potential effectiveness of these treatments to reduce almond allergenicity.

FPCount protocol - Short protocol v1

2021 ◽  
Author(s):  
Eszter Csibra ◽  
Guy-Bart Stan
Keyword(s):  
Protein Concentration ◽  
Fluorescent Protein ◽  
R Package ◽  
Protein Quantification ◽  
Short Version ◽  
Protein Storage ◽  
Concentration Determination ◽  
Sds Page ◽  
Plate Reader ◽  
Microplate Reader

FPCount is a complete protocol for fluorescent protein calibration, consisting of: 1. FP expression/purification using Thermo's HisPur Cobalt Resin. 2. FP concentration determination in a microplate reader. 3. FP fluorescence quantification in a microplate reader. Results can be analysed with the corresponding R package, FPCountR. This short version uses the ECmax protein quantification protocol, and is only suitable for FPs with entries in FPbase. If you want to verify or validate results, it's recommended you follow the complete protocol, which describes three protein quantification methods. The short protocol also skips the SDS-PAGE steps. If you require these, please see the complete protocol. --- Summary 1. Expression 2. Harvesting/Washing 3. Lysis 4. Fractionation 6. Purification 8. Protein concentration and buffer exchange 9. Quantification of FP concentration (part1) 10. Quantification of FP fluorescence 12. Protein storage 13. Calibration of Plate Reader

Sign in / Sign up

Export Citation Format

Share Document