PEG-4000 increased the mating efficiency of yeast-two hybrid screening process using PmF-box1 as bait

Protein degradation can occur through Ubiquitin 26S-Proteosome System (UPS). The degradation can be mediated by the SCF E3 ubiquitin ligase complex consisting of Skp1, Cullin, and F-box protein as the main components. The F-box protein at the C-terminal domain functions to recognize the targeted pro...

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Main Authors:	Nur Athirah Abd Hamid, Ismanizan Ismail
Format:	Article
Language:	English
Published:	Penerbit Universiti Kebangsaan Malaysia 2018
Online Access:	http://journalarticle.ukm.my/12916/ http://journalarticle.ukm.my/12916/ http://journalarticle.ukm.my/12916/1/04%20Nur%20Athirah%20Abd%20Hamid.pdf

id	ukm-12916
recordtype	eprints
spelling	ukm-129162019-05-15T10:58:02Z http://journalarticle.ukm.my/12916/ PEG-4000 increased the mating efficiency of yeast-two hybrid screening process using PmF-box1 as bait Nur Athirah Abd Hamid, Ismanizan Ismail, Protein degradation can occur through Ubiquitin 26S-Proteosome System (UPS). The degradation can be mediated by the SCF E3 ubiquitin ligase complex consisting of Skp1, Cullin, and F-box protein as the main components. The F-box protein at the C-terminal domain functions to recognize the targeted protein to be ubiquitinated and degraded via UPS. A stress-responsive F-box gene, PmF-box1 from Persicaria minor was categorized in the F-box containing kelch repeat (FBK) family; a family that specific to plant kingdom. To identify the targeted protein of PmF-box1, yeast-two hybrid system (Y2H) was used. In the Y2H screening process, mating efficiency is very important to fish out the interacting proteins. Therefore, one modification was conducted to increase the mating efficiency. In this screening, PmF-box1 was used as a bait to screen for the Y2H library which was constructed using RNA from plant samples treated with abscisic acid (ABA) and polyethylene glycol (PEG)-8000 and control sample. Autoactivation and toxicity tests of bait were performed before the Y2H screening. Tests on PmF-box1 showed that it is not toxic to the yeast and cannot autoactivate the yeast reporter genes. Mating efficiency was improved from 2.07% to 9.15% after addition of PEG-4000 in the mating culture compared to the original protocol, which it also increased the colony number in the screening step afterward. Additionally, bands of gene with different sizes were observed on electrophoresis gel after colony PCR analysis from the improved technique. Those genes may code for potential interacting proteins that needs further identification and confirmation. Penerbit Universiti Kebangsaan Malaysia 2018-12 Article PeerReviewed application/pdf en http://journalarticle.ukm.my/12916/1/04%20Nur%20Athirah%20Abd%20Hamid.pdf Nur Athirah Abd Hamid, and Ismanizan Ismail, (2018) PEG-4000 increased the mating efficiency of yeast-two hybrid screening process using PmF-box1 as bait. Sains Malaysiana, 47 (12). pp. 2961-2968. ISSN 0126-6039 http://www.ukm.my/jsm/malay_journals/jilid47bil12_2018/KandunganJilid47Bil12_2018.html
repository_type	Digital Repository
institution_category	Local University
institution	Universiti Kebangasaan Malaysia
building	UKM Institutional Repository
collection	Online Access
language	English
description	Protein degradation can occur through Ubiquitin 26S-Proteosome System (UPS). The degradation can be mediated by the SCF E3 ubiquitin ligase complex consisting of Skp1, Cullin, and F-box protein as the main components. The F-box protein at the C-terminal domain functions to recognize the targeted protein to be ubiquitinated and degraded via UPS. A stress-responsive F-box gene, PmF-box1 from Persicaria minor was categorized in the F-box containing kelch repeat (FBK) family; a family that specific to plant kingdom. To identify the targeted protein of PmF-box1, yeast-two hybrid system (Y2H) was used. In the Y2H screening process, mating efficiency is very important to fish out the interacting proteins. Therefore, one modification was conducted to increase the mating efficiency. In this screening, PmF-box1 was used as a bait to screen for the Y2H library which was constructed using RNA from plant samples treated with abscisic acid (ABA) and polyethylene glycol (PEG)-8000 and control sample. Autoactivation and toxicity tests of bait were performed before the Y2H screening. Tests on PmF-box1 showed that it is not toxic to the yeast and cannot autoactivate the yeast reporter genes. Mating efficiency was improved from 2.07% to 9.15% after addition of PEG-4000 in the mating culture compared to the original protocol, which it also increased the colony number in the screening step afterward. Additionally, bands of gene with different sizes were observed on electrophoresis gel after colony PCR analysis from the improved technique. Those genes may code for potential interacting proteins that needs further identification and confirmation.
format	Article
author	Nur Athirah Abd Hamid, Ismanizan Ismail,
spellingShingle	Nur Athirah Abd Hamid, Ismanizan Ismail, PEG-4000 increased the mating efficiency of yeast-two hybrid screening process using PmF-box1 as bait
author_facet	Nur Athirah Abd Hamid, Ismanizan Ismail,
author_sort	Nur Athirah Abd Hamid,
title	PEG-4000 increased the mating efficiency of yeast-two hybrid screening process using PmF-box1 as bait
title_short	PEG-4000 increased the mating efficiency of yeast-two hybrid screening process using PmF-box1 as bait
title_full	PEG-4000 increased the mating efficiency of yeast-two hybrid screening process using PmF-box1 as bait
title_fullStr	PEG-4000 increased the mating efficiency of yeast-two hybrid screening process using PmF-box1 as bait
title_full_unstemmed	PEG-4000 increased the mating efficiency of yeast-two hybrid screening process using PmF-box1 as bait
title_sort	peg-4000 increased the mating efficiency of yeast-two hybrid screening process using pmf-box1 as bait
publisher	Penerbit Universiti Kebangsaan Malaysia
publishDate	2018
url	http://journalarticle.ukm.my/12916/ http://journalarticle.ukm.my/12916/ http://journalarticle.ukm.my/12916/1/04%20Nur%20Athirah%20Abd%20Hamid.pdf
first_indexed	2023-09-18T20:03:41Z
last_indexed	2023-09-18T20:03:41Z
_version_	1777407016129527808

PEG-4000 increased the mating efficiency of yeast-two hybrid screening process using PmF-box1 as bait

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