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 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.

Protein quantification by bicinchoninic acid (BCA) assay follows complex kinetics and can be performed at short incubation times

2020 ◽  
Vol 608 ◽  
pp. 113904 ◽  
Author(s):  
Javiera Cortés-Ríos ◽  
Ana María Zárate ◽  
Juan David Figueroa ◽  
Joaquín Medina ◽  
Eduardo Fuentes-Lemus ◽  
...  
Keyword(s):  
Protein Quantification ◽  
Bicinchoninic Acid ◽  
Bca Assay

A simple Dot Blot Assay for population scale screening of DNA methylation

2018 ◽  
Author(s):  
Nelia Luviano ◽  
Sayuri Diaz-Palma ◽  
Céline Cosseau ◽  
Christoph Grunau
Keyword(s):  
Dna Methylation ◽  
Large Scale ◽  
General Rule ◽  
Enzyme Linked Immunosorbent Assay ◽  
Global Dna Methylation ◽  
Epigenetic Changes ◽  
Dot Blot ◽  
Wide Range ◽  
Dot Blot Assay ◽  
Experimental Populations

AbstractThe study of epigenetic changes in natural and experimental populations has increased the need to find a cost-effective and high throughput method to analyze multiple samples to effectuate a population-wide screening to study epigenetic changes triggered by biotic or abiotic stress. One of the most studied epigenetic marks is global DNA methylation, its measurement is used as a first step to differentiate methylation between individuals. There is a wide range of methods designed to detect genome-wide 5 methyl-cytosine (5mC) that differ in sensitivity, price, level of expertise required, but as a general rule, require large amounts of DNA and are relatively expensive. This is a limit for the analysis of 5mC in a large number of individuals as a prerequisite to population-wide testing of methylation markers. In this work, we evaluated a method based on antibody recognition of 5mC to measure the DNA methylation level of individuals of the species Biomphalaria glabrata, the intermediate host of schistosomiases, a neglected tropical disease. We validated the method to complete a large screening in the genome of B. glabrata snails treated with a chemical inhibitor of DNA methylation; however, the method can be applied to any species containing 5mC. The dot blot assay is a suitable method to perform a large-scale screening of global DNA methylation to compare 5mC levels between individuals from different natural or experimental populations. The dot blot method compares favorably with methods with an equivalent sensitivity such as the Enzyme Linked Immunosorbent Assay (ELISA) kit since it requires a smaller amount of DNA (30 ng) is less expensive and allows many more samples to be analyzed.

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

Extraction of DNA from a Wide Range of Objects by Treatment with Ammonium Salts

2020 ◽  
Vol 36 (6) ◽  
pp. 98-106
Author(s):  
E.I. Levitin ◽  
B.V. Sviridov ◽  
O.V. Piksasova ◽  
T.E. Shustikova
Keyword(s):  
Dna Extraction ◽  
Dna Isolation ◽  
Tissue Samples ◽  
New Approach ◽  
Cell Wall Disruption ◽  
Wide Range ◽  
Low Salt ◽  
Mammalian Blood ◽  
Plasmid Extraction ◽  
Two Hybrid

Currently, simple, rapid, and efficient techniques for DNA isolation from a wide range of organisms are in demand in biotechnology and bioinformatics. A key (and often limiting) step is the cell wall disruption and subsequent DNA extraction from the disintegrated cells. We have developed a new approach to DNA isolation from organisms with robust cell walls. The protocol includes the following steps: treatment of cells or tissue samples with ammonium acetate followed by cell lysis in low-salt buffer with the addition of SDS. Further DNA extraction is carried out according to standard methods. This approach is efficient for high-molecular native DNA isolation from bacteria, ascomycetes, yeast, and mammalian blood; it is also useful for express analysis of environmental microbial isolates and for plasmid extraction for two-hybrid library screening. express method for DNA isolation; ammonium salt treatment (в русских ключевых такой порядок), osmotic breakage of cells This study was financially supported by the NRC "Kurchatov Institute"-GOSNIIGENETIKA Kurchatov Genomic Center.

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.

The triiodothyronine uptake test: an assessment of methods.

1981 ◽  
Vol 27 (7) ◽  
pp. 1272-1276 ◽  
Author(s):  
L R Witherspoon ◽  
S E Shuler ◽  
M M Garcia
Keyword(s):  
Functional Status ◽  
Coefficient Of Variation ◽  
Temperature Effects ◽  
Thyroxine Binding Globulin ◽  
Wide Range ◽  
Free Thyroxine Index ◽  
Total Thyroxine ◽  
Multiple Measurements ◽  
Uptake Test ◽  
Commercial Kits

Abstract How well the free thyroxine index reflects thyroid functional status depends on the degree to which the triiodothyronine uptake test normalizes the effects of thyroxine binding protein concentrations on the total thyroxine concentration. We examined eight triiodothyronine uptake tests in which were used different secondary binders representative of those available in commercial kits. The relation between triiodothyronine uptake and thyroxine-binding globulin concentrations was established by use of sera from euthyroid individuals. We examined the effects of both high (greater than 20 mg/L) and low (less than 10 mg/L) thyroxine-binding globulin concentrations on triiodothyronine uptake. The precision of each assay, expressed as within- and between-run coefficient of variation, was calculated from multiple measurements on high, low, and midrange triiodothyronine uptake serum pools. The effects of variation in temperature and in exposure times were examined. The clinical most useful assays exhibited the ability to reflect a wide range of thyroxine-binding globulin concentrations and demonstrated little or no time or temperature effects.

Quantitating Apoptosis by a Nonradioactive DNA Dot Blot Assay

1994 ◽  
Vol 221 (2) ◽  
pp. 431-433 ◽  
Author(s):  
A.O. Hueber ◽  
M. Pierres ◽  
H.T. He
Keyword(s):  
Dot Blot ◽  
Dot Blot Assay

Laboratory Specimens and Genetic Privacy: Evolution of Legal Theory

2013 ◽  
Vol 41 (S1) ◽  
pp. 65-68 ◽  
Author(s):  
Michelle Huckaby Lewis
Keyword(s):  
Biomedical Research ◽  
Biological Samples ◽  
Biological Tissue ◽  
Genetic Information ◽  
Legal Theory ◽  
Genetic Privacy ◽  
Tissue Samples

Human biological tissue samples are an invaluable resource for biomedical research designed to find causes of diseases and their treatments. Controversy has arisen, however, when research has been conducted with laboratory specimens either without the consent of the source of the specimen or when the research conducted with the specimen has expanded beyond the scope of the original consent agreement. Moreover, disputes have arisen regarding which party, the researcher or the source of the specimen, has control over who may use the specimens and for what purposes. The purposes of this article are: (1) to summarize the most important litigation regarding the use of laboratory specimens, and (2) to demonstrate how legal theory regarding control of laboratory specimens has evolved from arguments based upon property interests in biological samples to claims that the origins of laboratory specimens have privacy interests in their genetic information that should be protected.

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