Extraction and Separation Analysis of Total Protein from the Bark of Apple Branches

doi:10.11983/CBB14186

Abstract

Abstract: We aimed to establish a suitable extraction method for analyzing total protein in the bark of apple branches. The branches of an 8-year-old cultivar (Huayue) were used as material. We compared 3 different extraction methods, including TCA-acetone precipitation (A), methanol/ammonium acetate precipitation (B), and improved Tris-phenol extraction (C). We optimized the extraction procedures and established an ideal method for analyzing the total protein in the bark of apple branches. The improved Tris-phenol extraction method could reveal a good map of 993 protein spots, significantly more than the other two methods: 418 and 674 protein spots were obtained with TCA-acetone precipitation and methanol/ammonium acetate precipitation, respectively. Furthermore, the gel background was clear, and the protein spots were better focused than with the two other methods. After comparing sample loading quantity, we selected 800 μg as the ideal loading quantity for our study. Furthermore, we selected several proteins for MALDI-TOF-TOF/MS analysis and database searching, and the protein spots were finally identified. In this study, we optimized the extraction procedures and sample loading quantity for total protein and finally obtained an ideal 2-D electrophoresis map. Our study may provide a good basis for further proteome analysis of apple branches.

Caixia Zhang, Yi Tian, Liyi Zhang, Long Xiao, Peihua Cong. Extraction and Separation Analysis of Total Protein from the Bark of Apple Branches[J]. Chinese Bulletin of Botany, 2015, 50(6): 739-745.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB14186

https://www.chinbullbotany.com/EN/Y2015/V50/I6/739

Figures/Tables 4

References 22

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Spot No.	Protein name	Accession No.	Theoretical MW (kDa)/pI	Species	Protein score
1	ATP synthase subunit beta	gi\|470126069	60.12/6.01	Fragaria vesca	431
2	Actin	gi\|355329944	40.37/5.67	Malus domestica	587
3	Oxygen-evolving enhancer protein	gi\|470129332	35.35/6.1	Fragaria vesca	265
4	Oxygen-evolving enhancer protein 1chloroplastic-like	gi\|470129332	35.35/6.1	Fragaria vesca	569
5	Uncharacterized protein At2g37660	gi\|470133785	35.44/5.86	Fragaria vesca	91
6	PRUPE_ppa007855mg	gi\|462397824	37.65/8.09	Prunus persica	62
7	PRUPE_ppa006513mg	gi\|462411618	42.18/5.82	Prunus persica	289
8	Acetyl-CoA carboxylase 1 isoform 3	gi\|508724180	24.65/6.17	Theobroma cacao	64
9	Cytosolic malate dehydrogenase	gi\|78216493	35.97/6.01	Malus domestica	334
10	PRUPE_ppa007772mg	gi\|462411755	39.12/5.88	Prunus persica	222

Spot No.	Protein name	Accession No.	Theoretical MW (kDa)/pI	Species	Protein score
1	ATP synthase subunit beta	gi\|470126069	60.12/6.01	Fragaria vesca	431
2	Actin	gi\|355329944	40.37/5.67	Malus domestica	587
3	Oxygen-evolving enhancer protein	gi\|470129332	35.35/6.1	Fragaria vesca	265
4	Oxygen-evolving enhancer protein 1chloroplastic-like	gi\|470129332	35.35/6.1	Fragaria vesca	569
5	Uncharacterized protein At2g37660	gi\|470133785	35.44/5.86	Fragaria vesca	91
6	PRUPE_ppa007855mg	gi\|462397824	37.65/8.09	Prunus persica	62
7	PRUPE_ppa006513mg	gi\|462411618	42.18/5.82	Prunus persica	289
8	Acetyl-CoA carboxylase 1 isoform 3	gi\|508724180	24.65/6.17	Theobroma cacao	64
9	Cytosolic malate dehydrogenase	gi\|78216493	35.97/6.01	Malus domestica	334
10	PRUPE_ppa007772mg	gi\|462411755	39.12/5.88	Prunus persica	222