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ELISA based quantification of Pax6 expression in the developing Zebrafish embryos.

Kannan RR, Vincent SG - Ann Neurosci (2015)

Bottom Line: The purity was confirmed by SDS-PAGE and western blotting using Pax6 mouse monoclonal antibody.The zebrafish Pax6 protein was detected as 48 kDa and confirmed by western blotting.This study paves way to quantify the level of expression of proteins or transcription factors during early embryonic and larval development or embryogenesis using zebrafish as model system.

View Article: PubMed Central - PubMed

Affiliation: International Centre for Nanobiotechnology (ICN), Centre for Marine Science and Technology (CMST), Manonmaniam Sundaranar University, Rajakkamangalam, Kanyakumari Dist-629502, Tamil Nadu, India ; Centre for Nanoscience and Nanotechnology (CNSNT), Sathyabama University, Jeppiaar Nagar, Chennai - 600119, Tamil Nadu, INDIA.

ABSTRACT

Background: Transcription factors are the key regulators of metabolic pathways in the cells, tissues and organ development during embryogenesis. Pax6 is a transcription factor involved in vertebrate eye, brain and central nervous system formation during development.

Purpose: A reliable and sensitive assay for the spatiotemporal expression, quantification and detection of Pax6 is not available so far in zebrafish as a developmental model, hence the objective of this work is to develop quantitative assay in zebrafish embryos.

Methods: The Pax6 transcription factor was purified by heparin agarose affinity chromatography and DEAE cellulose chromatography techniques from the developing zebrafish embryos. The purity was confirmed by SDS-PAGE and western blotting using Pax6 mouse monoclonal antibody. The standard graph was plotted for Pax6 and the expressions in seventeen developmental stages were quantified by indirect ELISA.

Results: The maximum expression of Pax6 was detected at 8 hpf (hours post fertilization) and it was quantified as 179 ng/embryo from the average total protein of 9.5 µg/embryo. The zebrafish Pax6 protein was detected as 48 kDa and confirmed by western blotting.

Conclusion: This study paves way to quantify the level of expression of proteins or transcription factors during early embryonic and larval development or embryogenesis using zebrafish as model system.

No MeSH data available.


Related in: MedlinePlus

SDS- PAGE analysis of purified Pax6 (ELISA andWB confirmed).
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fig_2: SDS- PAGE analysis of purified Pax6 (ELISA andWB confirmed).

Mentions: Pax6 purification from zebrafish embryos were carried out by three steps of chromatography (affinity, ion exchange and gel filtration techniques). The total protein extracts were prepared from 8 hpf (hours post fertilization) of the zebrafish embryos for 100 mg in one mL (100 embryos). ELISA results revealed high level of Pax6 expression at 8 hpf, since the total protein for Pax6 purification was taken from this stage. Pax6 is purified using heparin agarose affinity chromatography and the obtained absorbance values are shown in Fig. 1a. The first elution showed more immunoreactivity in ELISA and hence it was concentrated by ammonium sulfate precipitation and followed by dialysis. The elution was purified in DEAE cellulose chromatography and the absorbance values are shown in Fig. 1b. Fraction D3 from ion exchange showed maximum level of Pax6 activity and this fraction was purified through gel filtration, in which, the elution G6 showed more immunoreactivity in ELISA (Fig. 1c) which confirmed the presence of Pax6. Analysis of SDS PAGE showed a single band with 48 kDa (Fig. 2), which implies a purified protein and this confirms the purity of Pax6 and confirmed by western blotting. The known quantity of this protein had established through BCA assay and was used as standard to plot the standard graph of Pax6 for ELISA. The linearity plot of the concentration dependent activity profile was plotted in Fig. 3.


ELISA based quantification of Pax6 expression in the developing Zebrafish embryos.

Kannan RR, Vincent SG - Ann Neurosci (2015)

SDS- PAGE analysis of purified Pax6 (ELISA andWB confirmed).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4481557&req=5

fig_2: SDS- PAGE analysis of purified Pax6 (ELISA andWB confirmed).
Mentions: Pax6 purification from zebrafish embryos were carried out by three steps of chromatography (affinity, ion exchange and gel filtration techniques). The total protein extracts were prepared from 8 hpf (hours post fertilization) of the zebrafish embryos for 100 mg in one mL (100 embryos). ELISA results revealed high level of Pax6 expression at 8 hpf, since the total protein for Pax6 purification was taken from this stage. Pax6 is purified using heparin agarose affinity chromatography and the obtained absorbance values are shown in Fig. 1a. The first elution showed more immunoreactivity in ELISA and hence it was concentrated by ammonium sulfate precipitation and followed by dialysis. The elution was purified in DEAE cellulose chromatography and the absorbance values are shown in Fig. 1b. Fraction D3 from ion exchange showed maximum level of Pax6 activity and this fraction was purified through gel filtration, in which, the elution G6 showed more immunoreactivity in ELISA (Fig. 1c) which confirmed the presence of Pax6. Analysis of SDS PAGE showed a single band with 48 kDa (Fig. 2), which implies a purified protein and this confirms the purity of Pax6 and confirmed by western blotting. The known quantity of this protein had established through BCA assay and was used as standard to plot the standard graph of Pax6 for ELISA. The linearity plot of the concentration dependent activity profile was plotted in Fig. 3.

Bottom Line: The purity was confirmed by SDS-PAGE and western blotting using Pax6 mouse monoclonal antibody.The zebrafish Pax6 protein was detected as 48 kDa and confirmed by western blotting.This study paves way to quantify the level of expression of proteins or transcription factors during early embryonic and larval development or embryogenesis using zebrafish as model system.

View Article: PubMed Central - PubMed

Affiliation: International Centre for Nanobiotechnology (ICN), Centre for Marine Science and Technology (CMST), Manonmaniam Sundaranar University, Rajakkamangalam, Kanyakumari Dist-629502, Tamil Nadu, India ; Centre for Nanoscience and Nanotechnology (CNSNT), Sathyabama University, Jeppiaar Nagar, Chennai - 600119, Tamil Nadu, INDIA.

ABSTRACT

Background: Transcription factors are the key regulators of metabolic pathways in the cells, tissues and organ development during embryogenesis. Pax6 is a transcription factor involved in vertebrate eye, brain and central nervous system formation during development.

Purpose: A reliable and sensitive assay for the spatiotemporal expression, quantification and detection of Pax6 is not available so far in zebrafish as a developmental model, hence the objective of this work is to develop quantitative assay in zebrafish embryos.

Methods: The Pax6 transcription factor was purified by heparin agarose affinity chromatography and DEAE cellulose chromatography techniques from the developing zebrafish embryos. The purity was confirmed by SDS-PAGE and western blotting using Pax6 mouse monoclonal antibody. The standard graph was plotted for Pax6 and the expressions in seventeen developmental stages were quantified by indirect ELISA.

Results: The maximum expression of Pax6 was detected at 8 hpf (hours post fertilization) and it was quantified as 179 ng/embryo from the average total protein of 9.5 µg/embryo. The zebrafish Pax6 protein was detected as 48 kDa and confirmed by western blotting.

Conclusion: This study paves way to quantify the level of expression of proteins or transcription factors during early embryonic and larval development or embryogenesis using zebrafish as model system.

No MeSH data available.


Related in: MedlinePlus