scholarly journals A Ponceau Staining based Dot-Blot Assay for reliable and cost-effective Protein Quantification

2019 ◽  
Author(s):  
Dario-Lucas Helbing ◽  
Leopold Böhm ◽  
Yan Cui ◽  
Leonie Karoline Stabenow ◽  
Helen Morrison
Keyword(s):  
Protein Extraction ◽  
Cost Effective ◽  
Protein Quantification ◽  
Dot Blot ◽  
Effective Manner ◽  
Dot Blot Assay ◽  
Ponceau S ◽  
Reliable Quantification ◽  
Complete Protein ◽  
Bca Assay

AbstractReliable quantification of protein extracts from tissues can be a challenge e.g. due to interference of the high fat content in tissues of the nervous system. Further problems like under- or overerstimation of protein concentrations in protein quantification kits like the bicinchoninic acid (BCA) assay can occur. In addition, common lysis buffers such as RIPA buffer are known to be unable to solubilize a large amount of proteins (~10-30%) leading to unsatisfactory and unreliable experimental results with techniques such as immunoblotting. In this work, we have developed a Ponceau S staining based protein quantification assay. This assay is compatible with tissues or cells directly lysed in 2x SDS gel loading buffer, containing bromophenolblue, leading to more complete protein extraction. Protein concentrations of several samples can be determined in a fast and cost-effective manner and subsequent experiments (e.g. Western blot) can be performed without loss of proteins. The presented protein quantification method is highly reliable, fast and economical. Using this method, it is possible to save between 2300 to 3200€ per 1000 lysates as compared to the costs of a commercial BCA kit.

scholarly journals A Ponceau S Staining-Based Dot Blot Assay for Rapid Protein Quantification of Biological Samples

Gels ◽  
2022 ◽  
Vol 8 (1) ◽  
pp. 43
Author(s):  
Dario Lucas Helbing ◽  
Leopold Böhm ◽  
Nova Oraha ◽  
Leonie Karoline Stabenow ◽  
Yan Cui
Keyword(s):  
Biological Samples ◽  
Protein Quantification ◽  
Dot Blot ◽  
Tissue Samples ◽  
Bicinchoninic Acid ◽  
Wide Range ◽  
Dot Blot Assay ◽  
Ponceau S ◽  
Commercial Kits ◽  
Bca Assay

Despite the availability of a wide range of commercial kits, protein quantification is often unreliable, especially for tissue-derived samples, leading to uneven loading in subsequent experiments. Here we show that the widely used Bicinchoninic Acid (BCA) assay tends to underestimate protein concentrations of tissue samples. We present a Ponceau S staining-based dot-blot assay as an alternative for protein quantification. This method is simple, rapid, more reliable than the BCA assay, compatible with biological samples lysed in RIPA or 2x SDS gel-loading buffer, and also inexpensive.

scholarly journals Quantification of Soluble or Insoluble Fractions of Leishmania Parasite Proteins in Microvolume Applications: A Simplification to Standard Lowry Assay

2020 ◽  
Vol 2020 ◽  
pp. 1-8 ◽  
Author(s):  
Bhagya Deepachandi ◽  
Sudath Weerasinghe ◽  
Thisira Priyantha Andrahennadi ◽  
Nadira D. Karunaweera ◽  
Nadeeja Wickramarachchi ◽  
...  
Keyword(s):  
Cost Effective ◽  
Accurate Method ◽  
Research Field ◽  
Protein Quantification ◽  
Color Intensity ◽  
Resource Limited Settings ◽  
Protein Assay ◽  
Resource Limited ◽  
Effective Manner ◽  
Lowry Assay

Protein quantification is often an essential step in any research field that involves proteins. Although the standard Lowry assay and its modifications are most abundantly used in protein quantification, the existing methods are rigid or often demonstrate nonlinearity between protein concentration and color intensity. A method for fast and accurate qualitative and/or quantitative determination of total soluble/insoluble proteins or micro-well plate immobilized proteins isolated from Leishmania parasites in microvolumes was described in the current study. Improvements in cost-effective techniques are necessary to increase the research outputs in resource-limited settings. This method is a modification to the established Lowry assay for protein quantification. Concentrations of unknown samples were calculated using a standard curve prepared using a standard series of bovine serum albumin (BSA). The optimized reagents were 2 N NaOH (sodium hydroxide), 2% Na2CO3 (sodium carbonate), 1% CuSO4 (copper sulfate), 2% KNaC4H4O6 (potassium sodium tartrate), and 2 N Folin and Ciocalteu’s phenol. This modified protein assay was sensitive for quantifying Leishmania proteins in a total crude extract or in a soluble fraction within the approximate range of 10–500 μg/ml (1–50 μg/assay) and showed a linearity between color intensity and concentration of the protein. This is an easier, fast, and accurate method for quantifying proteins with microvolumes in a cost-effective manner for routine use in research laboratories in resource-limited settings.

scholarly journals Rapid Screening of the Epidermal Growth Factor Receptor Phosphosignaling Pathway via Microplate-Based Dot Blot Assays

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Amedeo Cappione ◽  
Janet Smith ◽  
Masaharu Mabuchi ◽  
Timothy Nadler
Keyword(s):  
Large Scale ◽  
Solid Phase ◽  
Growth Factor Receptor ◽  
Cost Effective ◽  
Protein Microarrays ◽  
Rapid Screening ◽  
Dot Blot ◽  
A431 Cells ◽  
Dot Blot Assay ◽  
Multiple Samples

Expression profiling on a large scale, as is the case in drug discovery, is often accomplished through use of sophisticated solid-phase protein microarrays or multiplex bead technologies. While offering both high-throughput and high-content analysis, these platforms are often too cost prohibitive or technically challenging for many research settings. Capitalizing on the favorable attributes of the standard ELISA and slot blotting techniques, we developed a modified dot blot assay that provides a simple cost-effective alternative for semiquantitative expression analysis of multiple proteins across multiple samples. Similar in protocol to an ELISA, but based in a membrane bound 96-well microplate, the assay takes advantage of vacuum filtration to expedite the tedious process of washing in between binding steps. We report on the optimization of the assay and demonstrate its use in profiling temporal changes in phosphorylation events in the well-characterized EGF-induced signaling cascade of A431 cells.

Strategic planning in grassland farming: Principles andapplications

1997 ◽  
pp. 191-197
Author(s):  
W.J. Parker ◽  
N.M. Shadbolt ◽  
D.I. Gray
Keyword(s):  
Strategic Planning ◽  
Performance Indicators ◽  
Cost Effective ◽  
Key Performance Indicators ◽  
Strategic Plans ◽  
Effective Manner ◽  
Farm Business ◽  
Pastoral Farming ◽  
Financial Environment

Three levels of planning can be distinguished in grassland farming: strategic, tactical and operational. The purpose of strategic planning is to achieve a sustainable long-term fit of the farm business with its physical, social and financial environment. In pastoral farming, this essentially means developing plans that maximise and best match pasture growth with animal demand, while generating sufficient income to maintain or enhance farm resources and improvements, and attain personal and financial goals. Strategic plans relate to the whole farm business and are focused on the means to achieve future needs. They should be routinely (at least annually) reviewed and monitored for effectiveness through key performance indicators (e.g., Economic Farm Surplus) that enable progress toward goals to be measured in a timely and cost-effective manner. Failure to link strategy with control is likely to result in unfulfilled plans. Keywords: management, performance

APPLICATION OF REAL-TIME PRC TECHNIQUE AND REVERSE DOT-BLOT FOR DETECTION THE HIGH RISK TYPES OF HUMAN PAPILLOMAVIRUS CAUSING CERVICAL CANCER

2017 ◽  
pp. 99-103
Author(s):  
Van Bao Thang Phan ◽  
Hoang Bach Nguyen ◽  
Van Thanh Nguyen ◽  
Thi Nhu Hoa Tran ◽  
Viet Quynh Tram Ngo
Keyword(s):  
Cervical Cancer ◽  
High Risk ◽  
Real Time ◽  
Real Time Pcr ◽  
Dot Blot ◽  
The Real ◽  
Dot Blot Assay ◽  
Hpv Types ◽  
High Risk Hpv ◽  
Reverse Dot Blot

Introduction: Infection with HPV is the main cause of cervical cancer. Determining HPV infection and the types of HPV plays an important role in diagnosis, treatment and prognosis of cervicitis/cervical cancer. Aims: Determining proportion of high-risk HPV types and the occurrence of coinfection with multiple HPV types. Methods: 177 women with cervicitis or abnormal Pap smear result were enrolled in the study. Performing the real-time PCR for detecting HPV and the reverse DOT-BLOT assay for determining type of HPV in cases of positive PCR. Results: 7 types of high-risk HPV was dectected, the majority of these types were HPV type 18 (74.6%) and HPV type 16 (37.6%); the proportion of infection with only one type of HPV was 30.4% and coinfection with multiple HPV types was higher (69.6%), the coinfected cases with 2 and 3 types were dominated (32.2% and 20.3%, respectively) and the coinfected cases with 4 and 5 types were rare. Conclusion: Use of the real-time PCR and reverse DOT-BLOT assay can determine the high-risk HPV types and the occurrence of coinfection with multiple HPV types. Key words: HPV type, Reverse DOT-BLOT, real-time PCR,PCR, cervical cancer

Use of monoclonal and polyclonal antibodies to detect prostatic acid phosphatase by immunoblotting.

1986 ◽  
Vol 32 (10) ◽  
pp. 1832-1835 ◽  
Author(s):  
P C Patel ◽  
L Aubin ◽  
J Côte
Keyword(s):  
Monoclonal Antibodies ◽  
Acid Phosphatase ◽  
Western Blot ◽  
Polyclonal Antibodies ◽  
Prostatic Acid Phosphatase ◽  
The Other ◽  
Dot Blot ◽  
Western Blots ◽  
Dot Blot Assay ◽  
Polyclonal Antisera

Abstract We investigated two techniques of immunoblotting--the Western blot and the dot blot--for use in detecting prostatic acid phosphatase (PAP, EC 3.1.3.2). We used polyclonal antisera to human PAP, produced in rabbits by hyperimmunization with purified PAP, and PAP-specific monoclonal antibodies in the immunoenzymatic protocols. We conclude that PAP can be readily detected by Western blots with use of polyclonal antisera, but not with monoclonal antibodies. On the other hand, using a dot blot assay, we could easily detect PAP with both polyclonal and monoclonal antibodies.

scholarly journals Novel Regeneration Approach for Creating Reusable FO-SPR Probes with NTA Surface Chemistry

Nanomaterials ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 186
Author(s):  
Jia-Huan Qu ◽  
Karen Leirs ◽  
Remei Escudero ◽  
Žiga Strmšek ◽  
Roman Jerala ◽  
...  
Keyword(s):  
Surface Chemistry ◽  
Fluorescent Protein ◽  
Cost Effective ◽  
Nitrilotriacetic Acid ◽  
Antibody Fragments ◽  
Rapid Screening ◽  
Red Fluorescent Protein ◽  
Effective Manner ◽  
Surface Regeneration ◽  
Sensitivity Specificity

To date, surface plasmon resonance (SPR) biosensors have been exploited in numerous different contexts while continuously pushing boundaries in terms of improved sensitivity, specificity, portability and reusability. The latter has attracted attention as a viable alternative to disposable biosensors, also offering prospects for rapid screening of biomolecules or biomolecular interactions. In this context here, we developed an approach to successfully regenerate a fiber-optic (FO)-SPR surface when utilizing cobalt (II)-nitrilotriacetic acid (NTA) surface chemistry. To achieve this, we tested multiple regeneration conditions that can disrupt the NTA chelate on a surface fully saturated with His6-tagged antibody fragments (scFv-33H1F7) over ten regeneration cycles. The best surface regeneration was obtained when combining 100 mM EDTA, 500 mM imidazole and 0.5% SDS at pH 8.0 for 1 min with shaking at 150 rpm followed by washing with 0.5 M NaOH for 3 min. The true versatility of the established approach was proven by regenerating the NTA surface for ten cycles with three other model system bioreceptors, different in their size and structure: His6-tagged SARS-CoV-2 spike fragment (receptor binding domain, RBD), a red fluorescent protein (RFP) and protein origami carrying 4 RFPs (Tet12SN-RRRR). Enabling the removal of His6-tagged bioreceptors from NTA surfaces in a fast and cost-effective manner can have broad applications, spanning from the development of biosensors and various biopharmaceutical analyses to the synthesis of novel biomaterials.

scholarly journals Postnatal management of children with antenatal hydronephrosis

2020 ◽  
Vol 26 (1) ◽  
Author(s):  
Mohammed S. ElSheemy
Keyword(s):  
Renal Function ◽  
Urinary Tract Obstruction ◽  
Cost Effective ◽  
Voiding Cystourethrography ◽  
Main Body ◽  
Antenatal Hydronephrosis ◽  
Postnatal Management ◽  
Effective Manner ◽  
Lower Urinary Tract Obstruction

Abstract Background Postnatal management of infants with antenatal hydronephrosis (ANH) is still one of the most controversial issues. The majority of infants with ANH are asymptomatic with only few children who develop renal insufficiency. Thus, the biggest challenge for pediatric urologists is to distinguish children who will require further investigations and possible intervention prior to the development of symptoms, complications or renal damage in a cost effective manner without exposing them to the hazards of unnecessary investigations. Main body In this review article, literature on ANH were reviewed to present the current suggestions, recommendations, guidelines and their rational for postnatal management of ANH. It is agreed that a large portion of infants with ANH will improve; thus, the protocol of management is based mainly on observation and follow-up by ultrasound to detect either resolution, stabilization or worsening of hydronephrosis. The first 2 years of life are critical for this follow-up as the final picture is mostly reached during that period. Advanced imaging using voiding cystourethrography or renal scintigraphy are required for children at risk. Then, surgical intervention is selected only for a subgroup of these infants who showed worsening of hydronephrosis or renal function. Conclusions The protocol of management is based mainly on observation and follow-up by US to detect either resolution, stabilization or worsening of hydronephrosis. Postnatal evaluation should be performed for any neonate with a history ANH at any stage during pregnancy even if it was resolved during third trimester. Exclusion of UTI should be performed by urinalysis for all cases followed by urine culture if indicated. Serum creatinine should be performed especially in patients with bilateral ANH. US is the initial standard diagnostic imaging technique. Other imaging modalities like VCUG and nuclear renal scans may be required according to the results of the US evaluation. The most important items in decision making are the presence of bilateral or unilateral hydronephrosis, presence or absence of hydroureter, presence of lower urinary tract obstruction and degree of hydronephrosis on the initial postnatal US. Then an intervention is selected only for a subgroup of these patients who showed deterioration in renal function or degree of hydronephrosis or were complicated by UTIs. All these recommendations are based on the available literature. However, management of ANH is still a controversial issue due to lack of high evidence-based recommendations. Randomised controlled studies are still needed to provide a high level evidence for different aspects of management.

scholarly journals An Artificial-Intelligence-Discovered Functional Ingredient, NRT_N0G5IJ, Derived from Pisum sativum, Decreases HbA1c in a Prediabetic Population

Nutrients ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 1635
Author(s):  
Sweeny Chauhan ◽  
Alish Kerr ◽  
Brian Keogh ◽  
Stephanie Nolan ◽  
Rory Casey ◽  
...  
Keyword(s):  
Artificial Intelligence ◽  
Pisum Sativum ◽  
Cost Effective ◽  
Glycated Haemoglobin ◽  
Glucose Regulation ◽  
Functional Ingredient ◽  
Effective Manner ◽  
Human Skeletal Muscle Cells ◽  
Increased Risk

The prevalence of prediabetes is rapidly increasing, and this can lead to an increased risk for individuals to develop type 2 diabetes and associated diseases. Therefore, it is necessary to develop nutritional strategies to maintain healthy glucose levels and prevent glucose metabolism dysregulation in the general population. Functional ingredients offer great potential for the prevention of various health conditions, including blood glucose regulation, in a cost-effective manner. Using an artificial intelligence (AI) approach, a functional ingredient, NRT_N0G5IJ, was predicted and produced from Pisum sativum (pea) protein by hydrolysis and then validated. Treatment of human skeletal muscle cells with NRT_N0G5IJ significantly increased glucose uptake, indicating efficacy of this ingredient in vitro. When db/db diabetic mice were treated with NRT_N0G5IJ, we observed a significant reduction in glycated haemoglobin (HbA1c) levels and a concomitant benefit on fasting glucose. A pilot double-blinded, placebo controlled human trial in a population of healthy individuals with elevated HbA1c (5.6% to 6.4%) showed that HbA1c percentage was significantly reduced when NRT_N0G5IJ was supplemented in the diet over a 12-week period. Here, we provide evidence of an AI approach to discovery and demonstrate that a functional ingredient identified using this technology could be used as a supplement to maintain healthy glucose regulation.

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