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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.


Workflow’s chart. (A) Graphic representation of our protocol; All four fractions extracted are assayed in a Bradford reaction to estimate the total yield of proteins extracted for each extraction in Whole Larvae (WL) (B1) and in tissues (B2). CF: Cytoplasmic fraction; W1: supernatant obtained after washing with WASH150 solution; W2: supernatant obtained after washing with WASH250 solution; NF: Nuclear fraction. Standard error is obtained from independent mensuration of six different extractions; (C) 3 μg of NF and CF samples extracted from WL are loaded in 10% acrylamide gel to monitor fractioning of the protein extraction procedures.
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ijms-16-12360-f001: Workflow’s chart. (A) Graphic representation of our protocol; All four fractions extracted are assayed in a Bradford reaction to estimate the total yield of proteins extracted for each extraction in Whole Larvae (WL) (B1) and in tissues (B2). CF: Cytoplasmic fraction; W1: supernatant obtained after washing with WASH150 solution; W2: supernatant obtained after washing with WASH250 solution; NF: Nuclear fraction. Standard error is obtained from independent mensuration of six different extractions; (C) 3 μg of NF and CF samples extracted from WL are loaded in 10% acrylamide gel to monitor fractioning of the protein extraction procedures.

Mentions: Solution and glassware are pre-chilled on ice before use and in order to minimize protein degradation, all steps in this protocol have been carried on ice. About 20 to 30 third instar WL (or 60–70 couple of SG, 60 B or 60 MT tissues), grown on standard corn medium at 25 °C, are transferred on petri capsule, washed with NaCl 0.7% and then transferred on 1.5 mL Eppendorf tube. Larvae are bulk-homogenized with a sterile Polypropylene (PP) pestle in 200 μL of Cytoplasmic Extraction Buffer 1X (CytoEB1X). The ice-cold homogenate is then centrifuged to spin down ghost larvae and the supernatant is carefully removed and processed in a further steps (Sur0) (Figure 1A). Optionally about 100 μL of CytoEB1X could be added on slurry ghost larvae and re-homogenized for a better proteins yield. About 150–200 μL of supernatant is centrifuged and the obtained supernatant is carefully recovered and clarified with a second centrifugation step obtaining thus Cytoplasmic Fraction (CF). The dried pellet is used for nuclear proteins extraction in further purification steps. To ensure that cells are completely lysed, two washing steps are performed with increased sucrose concentration using the two wash buffers WASH150 and WASH250. One hundred and fifty microliters of WASH150 are used to re-suspend pellet up and down avoiding bubbles formation. This solution is centrifuged and the obtained supernatant is carefully recovered and clarified with a second centrifugation step, keeping aside Wash1 (W1).


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)

Workflow’s chart. (A) Graphic representation of our protocol; All four fractions extracted are assayed in a Bradford reaction to estimate the total yield of proteins extracted for each extraction in Whole Larvae (WL) (B1) and in tissues (B2). CF: Cytoplasmic fraction; W1: supernatant obtained after washing with WASH150 solution; W2: supernatant obtained after washing with WASH250 solution; NF: Nuclear fraction. Standard error is obtained from independent mensuration of six different extractions; (C) 3 μg of NF and CF samples extracted from WL are loaded in 10% acrylamide gel to monitor fractioning of the protein extraction procedures.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490448&req=5

ijms-16-12360-f001: Workflow’s chart. (A) Graphic representation of our protocol; All four fractions extracted are assayed in a Bradford reaction to estimate the total yield of proteins extracted for each extraction in Whole Larvae (WL) (B1) and in tissues (B2). CF: Cytoplasmic fraction; W1: supernatant obtained after washing with WASH150 solution; W2: supernatant obtained after washing with WASH250 solution; NF: Nuclear fraction. Standard error is obtained from independent mensuration of six different extractions; (C) 3 μg of NF and CF samples extracted from WL are loaded in 10% acrylamide gel to monitor fractioning of the protein extraction procedures.
Mentions: Solution and glassware are pre-chilled on ice before use and in order to minimize protein degradation, all steps in this protocol have been carried on ice. About 20 to 30 third instar WL (or 60–70 couple of SG, 60 B or 60 MT tissues), grown on standard corn medium at 25 °C, are transferred on petri capsule, washed with NaCl 0.7% and then transferred on 1.5 mL Eppendorf tube. Larvae are bulk-homogenized with a sterile Polypropylene (PP) pestle in 200 μL of Cytoplasmic Extraction Buffer 1X (CytoEB1X). The ice-cold homogenate is then centrifuged to spin down ghost larvae and the supernatant is carefully removed and processed in a further steps (Sur0) (Figure 1A). Optionally about 100 μL of CytoEB1X could be added on slurry ghost larvae and re-homogenized for a better proteins yield. About 150–200 μL of supernatant is centrifuged and the obtained supernatant is carefully recovered and clarified with a second centrifugation step obtaining thus Cytoplasmic Fraction (CF). The dried pellet is used for nuclear proteins extraction in further purification steps. To ensure that cells are completely lysed, two washing steps are performed with increased sucrose concentration using the two wash buffers WASH150 and WASH250. One hundred and fifty microliters of WASH150 are used to re-suspend pellet up and down avoiding bubbles formation. This solution is centrifuged and the obtained supernatant is carefully recovered and clarified with a second centrifugation step, keeping aside Wash1 (W1).

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.