From 729aa6436048b081ab3678c87fef85d11b14a1f3 Mon Sep 17 00:00:00 2001 From: Christopher Lux <lux@csbiology.de> Date: Mon, 8 Apr 2024 12:56:28 +0200 Subject: [PATCH] moved elements from study to assays --- assays/ChloroplasMembraneEnrichment/README.md | 0 .../dataset/.gitkeep | 0 .../isa.assay.xlsx | Bin 0 -> 10684 bytes .../protocols/.gitkeep | 0 .../Enrichment of envelope membranes.md | 4 ++++ assays/ChloroplastIsolation/README.md | 0 assays/ChloroplastIsolation/dataset/.gitkeep | 0 assays/ChloroplastIsolation/isa.assay.xlsx | Bin 0 -> 11741 bytes .../ChloroplastIsolation/protocols/.gitkeep | 0 ...ation of Chloroplast Envelope Membranes.md | 6 ++++++ assays/ColdChloroEnvelope/isa.assay.xlsx | Bin 21086 -> 21086 bytes isa.investigation.xlsx | Bin 7353 -> 7353 bytes studies/ColdChloroEnvelope/isa.study.xlsx | Bin 22192 -> 14386 bytes 13 files changed, 10 insertions(+) create mode 100644 assays/ChloroplasMembraneEnrichment/README.md create mode 100644 assays/ChloroplasMembraneEnrichment/dataset/.gitkeep create mode 100644 assays/ChloroplasMembraneEnrichment/isa.assay.xlsx create mode 100644 assays/ChloroplasMembraneEnrichment/protocols/.gitkeep create mode 100644 assays/ChloroplasMembraneEnrichment/protocols/Enrichment of envelope membranes.md create mode 100644 assays/ChloroplastIsolation/README.md create mode 100644 assays/ChloroplastIsolation/dataset/.gitkeep create mode 100644 assays/ChloroplastIsolation/isa.assay.xlsx create mode 100644 assays/ChloroplastIsolation/protocols/.gitkeep create mode 100644 assays/ChloroplastIsolation/protocols/Isolation of Chloroplast Envelope Membranes.md diff --git a/assays/ChloroplasMembraneEnrichment/README.md b/assays/ChloroplasMembraneEnrichment/README.md new file mode 100644 index 0000000..e69de29 diff --git a/assays/ChloroplasMembraneEnrichment/dataset/.gitkeep b/assays/ChloroplasMembraneEnrichment/dataset/.gitkeep new file mode 100644 index 0000000..e69de29 diff --git a/assays/ChloroplasMembraneEnrichment/isa.assay.xlsx b/assays/ChloroplasMembraneEnrichment/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..2156966f91bb7aa7d211fc7aa8b9969206507505 GIT binary patch literal 10684 zcmai4bzGIp(?`0yQ@Xn)rMsnD={j_GgLFxEm(mTQbhng99weoakoO?h>%H82fA9Wr z&hvSm`R>fl?C#9Yj*=_{Bs>@x7%Ui=MwlkjD3HSP2^iQV6c`u==&g>Zovo9Jt&@S8 zJHW(IkIBu(x<_ruwwndD?Sh`nK-u_<(hDqqQHvNxm9H=iM<JR#4JPSxE3PkT%IKbo zi0x38f2w7#weplq(5s9`dd3_zI{$RH7Sh=$XnH8adt_yjI{6Yz+m8+3u|aZ-YCUwq z%Wi@?vBG<_40Vqk*irHpIvX!C%|&M2683aAc}e750Ohe!=UqQSKEzIJNJwCmq$-0^ zrO0th{$?Or=wR)}CYW`d1N1DNmT-=D$HZEUjE7o34ZE=yW#u87-%&06$f$|`%g->* zz0&>zh`Mf}X-Zzjm$OYX-EUsFWNy5cRZXrDHi~eTr%i1{etrqhI!zbJTK;@TztsoQ zllVi<OLFQT1m1{u1ClwOp`nMjm0=u=0fKv2S5+}W@eQ;ZUzEI{B~j`eAv&8EwUH6n zJYn?=I;A`|zuF$C<=>PBGQMyx6%H89c$X!S^kK35I#pcW#WEaLFLo~Gyg+K&wGks- z9}d&3&Yf^+-A?Hb(}3&%G7aeW&jt;8GH@`lc4YoV^I*Q?Z0Uuspl`C^U|<M;(lFgq zR>r?q_`rfFeCQ><ah+x>1)E#g`m|t~ibJrA!j_;uD*t2P!C`o;Fk+MiOtk$T&}?XT zgg<I0O68mok)a$P6KZHQbhK;ehlM=~9RhSHISd=105N^b^j3~srgWe^Mhi=-AKD}C zGRL_=K>iJVH0Ge<kc9~C*!0&hm#*m3vZ_O;s{y~Qpwfm>;e{5;l3UhEPHS5oyRUKk zMtM)ov~LD!Uda)K`@2scW`t;L<<cpQ1NGGBT5kl3v!upza~d+t(Ns$n)`qk@oe3B9 z;DBSVWknkAnhDc_1IS&q;9lOaO;U4@dx8DZ2x$0^v<3ls<!1e-%Kk|J_dxJ(n*Grc z7k>o$ECK2S|F2}Ob`Dm?c6L_xOXH`a+v7(pKd_(%9eTsRyU=d97Nw1Iqz%^9xp)#( z-yml#ZQdy4b6tjHLkL?0GnMcLi|>SW()5}Yc@2sD>@zJ*4IyMpZ~A9vc>SHeAwJZP z3wQ!<q3~1`pVV>EsR=oXiE6x`#>o-Vz8zlGm$77sPGch(FHE29Q+AAhm++k1b2*pc z<VL->8Agpd%h(L_V<xTev}#wU+jYY}f!DW@-LU8}F{w2RshhmnccSWmajoMg?t&e< z>{(Mx#8aJ{Mw$$)x|Q1gGiW31eWzU~R9owmZ+1D2K6@`Eh;NYhA8sRX5=D+I-LT7j zhagw%@I8b3Z-pa2DC%BCj^-vNPLHQ+V}x=`5NN7)gA|YZ589s))_c7sB`e#dv!ME| zT+qj^5b$tQkb;-FDA!A;bJ%2_@8M!pfJ-zaG+dvx^2lyf<;<YO@el|iUw34jqywv> zkVObeuL8OlNaMDf%U*TT_m72Mmt%#$Hu3J!o{0=-gB7(MMERaD8B>JTEdjeN{~X^c z)R~arb69k0D!y0@d;KI)E$)t<H8i8<Ch^X~8>#`agExBv;csUTjY?XQb*(X1T=N>K z!>}1^-*i}P7>?FOcf!oE-Zqg`HSIu)S)0)BM+U~1tnZGs>RU9}9MxtUjb5hi2XJ31 zI8?qa+eftb#qho!(aPwNqC1gU!y(iVn9O79XRJ7+iESEQ9=Tu|uzOY}HgaBhTa_gU z*KEabpe#dH-&ncVgJX}%sDMS`E^w|~Z#!(#Ql-z;4bZI)DEPXGMPRCxYITOWItX!a z+!cKdYlZ91lsbW#u+`^jcLYtyhD$O?5rbkv?07@E)16PszjI`m;Y!rqdhX_w!Hu#y zBJlZ)hZ$+Lkl0ReR43>gHZe01kWti4Hl`65SSjL0AJa$(G$D@p5tHKHHWLioI=E@J zzR`<?#PVFz0E#t>z7gsWW>G#uEkJE^Fwfg2?I-)j84%tQ`ZTVK8;(kbBtRG3fa*<& z59}nISGJ)D7%UsSSD|6|6WDlouX009|KWhx4t^FR6r`HatMl<vQ?yqoXZyK#3mBg5 zHimphNnbPf<fkJR`~l>rr7<m5<<PFdFX~>*mK_jsergC7lqh*#nJ55r6n-uG=@yli zk@dOj6-|Fm-He>J7jB;~eTF%A;73!@TK5LcNyKKi>6-W$Ib1@_J|Sw|ciIauXR_)I z_L=j+Dd)b!Pdf-Z2Zesk@`sJq+{-;q1GEcIG5_bxW&1gE7xWy{v@t`Eu1{zQ4+xe! zauwu?Ee8D|BJzftiz47uv0$VYvJ`#8EL3Kx1XT~fh33#3@3@SZtAu{=?CB`lCsTj5 zw=5}-o`{G^cS<KbES6|$5PbbwU0<xvrcN>DX!;Tl*xQ%hS=CuQ-*`hB9jQM5<&9!g zXdaLJ!oJrDDoNfAoj^~Yii?FySi~_)s~xbIA-VyW_n~8X-zj8YG?6eW1vz0)(W9VM zuO|F(a%7!U5Sv-P#*GAPa}$U%K<m5j^65spRW#Atli&MOL8$8dnpKTrL^iNUV)tz- z&qW_cWXHi!%&q8I{)pA#$@9uv()m{wSqBm2HOtiqmA+f{o=Z%8%er<6#~A$89`uo} zs_O}9L4zC%+;c!29blyHt4f=;Na73eSf9rAjUTx*o%#L~G9;1S4zJrUX+NiMa6UU; 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The intact chloroplast fraction was vigorously resuspended in 2 mL of buffer medium (10 mM MOPS-NaOH, pH 7.8) and kept on ice for 10 min to allow osmotic disruption of chloroplasts. To prevent protease-driven protein degradation the buffer medium was complemented with a protease inhibitor cocktail (cOmplete, EDTA-free, Sigma Aldrich; www.sigmaaldrich.com). At this step, 100-μL samples were collected from the lysate for the MS-based identification of total chloroplast proteins. + +A three-step Suc gradient (bottom to top: 4 mL 0.93 M, 0.6 M, and 0.3 M Suc) prepared in Ultra-Clear tubes (16 × 102 mm, Beckman Coulter; www.beckmann.de) was overlaid with 1 mL of disrupted chloroplast preparation. After ultra-centrifugation (70,000g for 1 h at 4°C, with no brake, on a swing-out rotor SureSpin 630, Thermo Fisher Scientific; www.thermo-fisher.com) the yellowish envelope fraction was collected from the interphase between 0.93 M and 0.6 M Suc (Supplemental Fig. S1). This fraction was 2× diluted with double distilled water and the envelope membranes were collected by ultra-centrifugation (400,000g for 20 min at 4°C on a ST120AT rotor, Thermo Fisher Scientific; www.thermofisher.com,). The resulting membrane fraction was resuspended in 1 mL double distilled water to remove any remaining Suc. To remove membrane-associated proteins the envelope membranes were resuspended in 1 M of sodium carbonate (Na2CO3) medium and centrifuged again, as described above. 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z1)w+7UvANFv`316`cJ<pj{Y6(FK_Bk>^~3j-`F7(e<S+yNdFW4&r|0&IyTkAbpO77 X<)xq=wl6FI7T^gedAp4I9{m3SfU?2* literal 0 HcmV?d00001 diff --git a/assays/ChloroplastIsolation/protocols/.gitkeep b/assays/ChloroplastIsolation/protocols/.gitkeep new file mode 100644 index 0000000..e69de29 diff --git a/assays/ChloroplastIsolation/protocols/Isolation of Chloroplast Envelope Membranes.md b/assays/ChloroplastIsolation/protocols/Isolation of Chloroplast Envelope Membranes.md new file mode 100644 index 0000000..c28a3b1 --- /dev/null +++ b/assays/ChloroplastIsolation/protocols/Isolation of Chloroplast Envelope Membranes.md @@ -0,0 +1,6 @@ +Isolation of Chloroplast Envelope Membranes + +The envelope membrane isolation procedure can be divided into two steps: (1) isolation of intact chloroplasts and (2) enrichment of envelope membranes from these chloroplasts using a Suc step gradient (Supplemental Fig. S1). The isolation of intact chloroplasts was carried out according to an existing protocol (Kunst, 1998) with some modifications: 200 g leaf material was chopped off 34-d-old Arabidopsis plants (cold acclimated and control plants kept at 22°C) and transferred to ice-cold homogenization buffer medium (0.45 M sorbitol, 20 mM Tricine-KOH, pH 8.4, 10 mM EDTA, 10 mM NaHCO3, and 0.1% [w/v] fatty-acid free bovine serum albumin). The ratio of buffer volume to weight of leaf material was 3:1 (v/w). In a glass beaker the buffer/leaf mixture was further cooled in ice water to limit metabolic activity to a minimum. After 30 min the mixture was transferred to a 1 L stainless steel beaker. For a controlled rupture of the leaves, the blender was successively run for 1 s at low, 1 s at medium, and 1 s at high settings (Waring commercial heavy-duty blender). This procedure was repeated twice. The disrupted leaf material was than filtered through three layers of Miracloth (http://www.merckmillipore.com), placed in a funnel, and the flow through collected in an ice-cooled Erlenmeyer flask. From this suspension the chloroplast fraction was collected by centrifugation (1,000g for 10 min at 4°C) and gently resuspended in 8 mL resuspension buffer medium (0.3 M sorbitol, 20 mM Tricine-KOH, pH 7.6, 5 mM MgCl2, and 2.5 mM EDTA) using a natural bristle paint brush. + +A Percoll gradient was prepared by mixing equal volumes of 2× concentrated resuspension buffer medium and pure Percoll. Of this mixture, 30 mL was transferred to 36-mL centrifuge tubes and centrifuged (Sorval SS34 fixed-angle rotor) at 43,400g for 30 min at 4°C with no brake. Two Percoll gradients were enough for 200 g of leaf material. The Percoll gradient was overlaid with the resuspended chloroplast suspension. After centrifugation in a HB4 swing-out rotor (13,300g for 15 min at 4°C, with no brake), two distinct green bands appeared (Supplemental Fig. S1). The upper band, containing broken chloroplasts, was removed using a water jet pump and the lower band was collected using a wide-open Pasteur pipette. This fraction was transferred to a SS34 tube and diluted with 3 volumes of resuspension buffer medium. 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