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Nuclear and Cytoplasmic Soluble Proteins Extraction from a Small Quantity of Drosophila's Whole Larvae and Tissues.

Lo Piccolo L, Bonaccorso R, Onorati MC - Int J Mol Sci (2015)

Bottom Line: The identification and study of protein's function in several model organisms is carried out using both nuclear and cytoplasmic extracts.For a long time, Drosophila's embryos have represented the main source for protein extractions, although in the last year, the importance of collecting proteins extracts also from larval tissues has also been understood.Here we report a very simple protocol, improved by a previously developed method, to produce in a single extraction both highly stable nuclear and cytoplasmic protein extracts from a small quantity of whole Drosophila's larvae or tissues, suitable for biochemical analyses like co-immunoprecipitation.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), Viale delle Scienze, Universita' degli Studi di Palermo, Palermo 90128, Italy. lucalopiccolo@gmail.com.

ABSTRACT
The identification and study of protein's function in several model organisms is carried out using both nuclear and cytoplasmic extracts. For a long time, Drosophila's embryos have represented the main source for protein extractions, although in the last year, the importance of collecting proteins extracts also from larval tissues has also been understood. Here we report a very simple protocol, improved by a previously developed method, to produce in a single extraction both highly stable nuclear and cytoplasmic protein extracts from a small quantity of whole Drosophila's larvae or tissues, suitable for biochemical analyses like co-immunoprecipitation.

No MeSH data available.


In vitro and in vivo analysis of proteins extracted from Drosophila’s small quantity of whole larva and tissues. Western Blot analysis of proteins extracted from WL to detect any contamination of cytoplasmic proteins in nuclear fractions and vice versa. The same amount (3 μg) of different protein fractions is used in an immunodetection assay with two different antibodies: anti-αH3 (1:4000) and anti-GAPDH (1:4000) (A1). The same extracts are used in an immunodetection assay with anti-AGO2 (1:2000) (A2); (B1) Western Blot analysis of proteins extracted from different Drosophila’s tissues like MT, SG and B. (B2) 3 μg of nuclear and cytoplasmic protein fractions are used in an immunodetection assay with anti-AGO2 (1:2000), anti-αH3 (1:4000), and anti-GAPDH (1:4000); (C) Co-immunoprecipitation of Squid and Hrb87F hnRNPs from nuclear extracts. The same amount of nuclear protein fractions is used in an immunoprecipitation assay with anti-Squid antibody and IgG. For the immunodetection assay anti-Squid (1:200) and anti-Hrb87F (1:4000) were used. I = Input, USquid = Unbound from anti-Squid IP, WSquid, wash from anti-Squid IP, UIgG = Unbound from IgG IP, WIgG, wash from anti-IgG IP, IPSquid = Immunoprecipitated material from anti-Squid, IP IPIgG = Immunoprecipitated material from IgG IP.
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ijms-16-12360-f002: In vitro and in vivo analysis of proteins extracted from Drosophila’s small quantity of whole larva and tissues. Western Blot analysis of proteins extracted from WL to detect any contamination of cytoplasmic proteins in nuclear fractions and vice versa. The same amount (3 μg) of different protein fractions is used in an immunodetection assay with two different antibodies: anti-αH3 (1:4000) and anti-GAPDH (1:4000) (A1). The same extracts are used in an immunodetection assay with anti-AGO2 (1:2000) (A2); (B1) Western Blot analysis of proteins extracted from different Drosophila’s tissues like MT, SG and B. (B2) 3 μg of nuclear and cytoplasmic protein fractions are used in an immunodetection assay with anti-AGO2 (1:2000), anti-αH3 (1:4000), and anti-GAPDH (1:4000); (C) Co-immunoprecipitation of Squid and Hrb87F hnRNPs from nuclear extracts. The same amount of nuclear protein fractions is used in an immunoprecipitation assay with anti-Squid antibody and IgG. For the immunodetection assay anti-Squid (1:200) and anti-Hrb87F (1:4000) were used. I = Input, USquid = Unbound from anti-Squid IP, WSquid, wash from anti-Squid IP, UIgG = Unbound from IgG IP, WIgG, wash from anti-IgG IP, IPSquid = Immunoprecipitated material from anti-Squid, IP IPIgG = Immunoprecipitated material from IgG IP.

Mentions: In order to detect any contamination of cytoplasmic proteins in nuclear fractions and vice versa, we checked the different fractions by Western Blot analysis loading 3 μg of protein extracts for each fraction. As shown in Figure 2A1, we obtained cytoplasmic fraction free of nuclear proteins and vice versa. We used two different antibodies, the anti-H3 to monitor proteins in the nuclear fraction and the anti-GAPDH to monitor proteins in the cytoplasmic fraction. In order to test the efficacy of our protocol, we performed a western blot analysis on the same proteins extracts using an antibody against AGO2 protein, a member of the Argonaute/PIWI protein family. As shown in Cernilogar et al. [11], AGO2 is present in both the nucleus and the cytoplasm. In extracts from WL the 100 kDa isoform is only present in the nuclear fraction, while the 80 kDa isoform is present in both cytoplasmic and nuclear fraction (Figure 2A2). Thus our protocol is optimal to monitor those proteins that move between nuclear and cytoplasmic compartment.


Nuclear and Cytoplasmic Soluble Proteins Extraction from a Small Quantity of Drosophila's Whole Larvae and Tissues.

Lo Piccolo L, Bonaccorso R, Onorati MC - Int J Mol Sci (2015)

In vitro and in vivo analysis of proteins extracted from Drosophila’s small quantity of whole larva and tissues. Western Blot analysis of proteins extracted from WL to detect any contamination of cytoplasmic proteins in nuclear fractions and vice versa. The same amount (3 μg) of different protein fractions is used in an immunodetection assay with two different antibodies: anti-αH3 (1:4000) and anti-GAPDH (1:4000) (A1). The same extracts are used in an immunodetection assay with anti-AGO2 (1:2000) (A2); (B1) Western Blot analysis of proteins extracted from different Drosophila’s tissues like MT, SG and B. (B2) 3 μg of nuclear and cytoplasmic protein fractions are used in an immunodetection assay with anti-AGO2 (1:2000), anti-αH3 (1:4000), and anti-GAPDH (1:4000); (C) Co-immunoprecipitation of Squid and Hrb87F hnRNPs from nuclear extracts. The same amount of nuclear protein fractions is used in an immunoprecipitation assay with anti-Squid antibody and IgG. For the immunodetection assay anti-Squid (1:200) and anti-Hrb87F (1:4000) were used. I = Input, USquid = Unbound from anti-Squid IP, WSquid, wash from anti-Squid IP, UIgG = Unbound from IgG IP, WIgG, wash from anti-IgG IP, IPSquid = Immunoprecipitated material from anti-Squid, IP IPIgG = Immunoprecipitated material from IgG IP.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4490448&req=5

ijms-16-12360-f002: In vitro and in vivo analysis of proteins extracted from Drosophila’s small quantity of whole larva and tissues. Western Blot analysis of proteins extracted from WL to detect any contamination of cytoplasmic proteins in nuclear fractions and vice versa. The same amount (3 μg) of different protein fractions is used in an immunodetection assay with two different antibodies: anti-αH3 (1:4000) and anti-GAPDH (1:4000) (A1). The same extracts are used in an immunodetection assay with anti-AGO2 (1:2000) (A2); (B1) Western Blot analysis of proteins extracted from different Drosophila’s tissues like MT, SG and B. (B2) 3 μg of nuclear and cytoplasmic protein fractions are used in an immunodetection assay with anti-AGO2 (1:2000), anti-αH3 (1:4000), and anti-GAPDH (1:4000); (C) Co-immunoprecipitation of Squid and Hrb87F hnRNPs from nuclear extracts. The same amount of nuclear protein fractions is used in an immunoprecipitation assay with anti-Squid antibody and IgG. For the immunodetection assay anti-Squid (1:200) and anti-Hrb87F (1:4000) were used. I = Input, USquid = Unbound from anti-Squid IP, WSquid, wash from anti-Squid IP, UIgG = Unbound from IgG IP, WIgG, wash from anti-IgG IP, IPSquid = Immunoprecipitated material from anti-Squid, IP IPIgG = Immunoprecipitated material from IgG IP.
Mentions: In order to detect any contamination of cytoplasmic proteins in nuclear fractions and vice versa, we checked the different fractions by Western Blot analysis loading 3 μg of protein extracts for each fraction. As shown in Figure 2A1, we obtained cytoplasmic fraction free of nuclear proteins and vice versa. We used two different antibodies, the anti-H3 to monitor proteins in the nuclear fraction and the anti-GAPDH to monitor proteins in the cytoplasmic fraction. In order to test the efficacy of our protocol, we performed a western blot analysis on the same proteins extracts using an antibody against AGO2 protein, a member of the Argonaute/PIWI protein family. As shown in Cernilogar et al. [11], AGO2 is present in both the nucleus and the cytoplasm. In extracts from WL the 100 kDa isoform is only present in the nuclear fraction, while the 80 kDa isoform is present in both cytoplasmic and nuclear fraction (Figure 2A2). Thus our protocol is optimal to monitor those proteins that move between nuclear and cytoplasmic compartment.

Bottom Line: The identification and study of protein's function in several model organisms is carried out using both nuclear and cytoplasmic extracts.For a long time, Drosophila's embryos have represented the main source for protein extractions, although in the last year, the importance of collecting proteins extracts also from larval tissues has also been understood.Here we report a very simple protocol, improved by a previously developed method, to produce in a single extraction both highly stable nuclear and cytoplasmic protein extracts from a small quantity of whole Drosophila's larvae or tissues, suitable for biochemical analyses like co-immunoprecipitation.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), Viale delle Scienze, Universita' degli Studi di Palermo, Palermo 90128, Italy. lucalopiccolo@gmail.com.

ABSTRACT
The identification and study of protein's function in several model organisms is carried out using both nuclear and cytoplasmic extracts. For a long time, Drosophila's embryos have represented the main source for protein extractions, although in the last year, the importance of collecting proteins extracts also from larval tissues has also been understood. Here we report a very simple protocol, improved by a previously developed method, to produce in a single extraction both highly stable nuclear and cytoplasmic protein extracts from a small quantity of whole Drosophila's larvae or tissues, suitable for biochemical analyses like co-immunoprecipitation.

No MeSH data available.