衣藻属（Chlamydomonas ）叶绿体蛋白质的35S脉冲标记

Alexandra-Viola Bohne; Christian Schwarz; Joerg Nickelsen

doi:10.21769/BioProtoc.783

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Peer-reviewed

³⁵S pulse Labelling of Chlamydomonas Chloroplast Proteins

衣藻属（Chlamydomonas ）叶绿体蛋白质的35S脉冲标记

AB Alexandra-Viola Bohne

CS Christian Schwarz

JN Joerg Nickelsen email

发布: 2013年06月05日第3卷第11期 DOI: 10.21769/BioProtoc.783 浏览次数: 10541

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Abstract

³⁵S pulse labelling of proteins is used to attach a radioactive label to newly synthesized proteins, as sulfur is an element that is mainly present in proteins (Fleischmann and Rochaix 1999). Depending on your organism’s uptake mechanisms you need cysteine, methionine or sulfuric acid as a source of radioactive sulfur. This example uses Chlamydomonas cells and H₂³⁵SO₄ (Schwarz et al., 2012).

Keywords: 35S pulse Labelling (35s脉冲标记)

Chlamydomonas (衣藻)

Protein Synthesis (蛋白质的合成)

Chloroplast Proteins (叶绿体蛋白)

Materials and Reagents

Strain of interest
Control strains lacking the gene for the proteins of interest (as a negative control)
Tris (Applichem, catalog number: A1379 )
Ammonium chloride (Carl Roth, catalog number: 5470 )
Magnesium chloride (Carl Roth, catalog number: KK36 )
Calcium chloride (Merck, catalog number: 1023780500 )
K₂HPO₄ (Applichem, catalog number: A1363 )
KH₂PO₄ (Applichem, catalog number: A1364 )
Na₂EDTA (Carl Roth, catalog number: 8043 )
ZnSO₄·7H₂O (Carl Roth, catalog number: T884 )
H₃BO₃ (Carl Roth, catalog number: P010 )
MnCl₂·4H₂O (Carl Roth, catalog number: 0 276 )
FeSO₄·7H₂O (Carl Roth, catalog number: P015 )
CoCl_2·6H₂O (Carl Roth, catalog number: T889 )
CuSO₄·5H₂O (Carl Roth, catalog number: 8175 )
(NH₄)₆Mo₇O_24·4H₂O (Carl Roth, catalog number: 3666 )
HEPES (Carl Roth, catalog number: HN78 )
Tricine (Carl Roth, catalog number: 6977 )
Methanol (Applichem, catalog number: A3493 )
Cycloheximide (Carl Roth, catalog number: 8682 )
³⁵S sulfuric acid (Hartmann Analytic, catalog number: S-RA-1 )
Liquid nitrogen (Linde, inquire)
Protease inhibitor cocktail (F. Hoffmann-La Roche, catalog number: 04693159001 )
TAP-B (see Recipes)
TAP-B/T(see Recipes)
Hutner trace elements (see Recipes)
Buffer A (see Recipes)
Buffer B (see Recipes)

Equipment

Sterile Erlenmeyer flasks (Brand KG)
Photometer (GE Healthcare)
Hemocytometer (Brand KG)
Microscope (≥ 400x magnification, Leica)
Reaction tubes (Sarstedt)
Screw cap microreaction tubes (Sarstedt)
Microreaction tube centrifuge with cooling capacity (Eppendorf)
Gel dryer (Bio-Rad Laboratories)
Whatman filter paper (GE Healthcare)

Procedure

English

中文翻译

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如何引用

Bohne, A., Schwarz, C. and Nickelsen, J. (2013). ³⁵S pulse Labelling of Chlamydomonas Chloroplast Proteins. Bio-protocol 3(11): e783. DOI: 10.21769/BioProtoc.783.