7. LAMPIRAN 7.1. Perhitungan efisiensi isolat proteinrepository.unika.ac.id/14666/8/12.70.0154...

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7. LAMPIRAN

7.1. Perhitungan efisiensi isolat protein

Isolat protein diperoleh dengan menggunakan presipitasi dengan 3 metode berbeda. Isolat

protein yang diambil berdasarkan perhitungan pada 3 batch dengan 3 kali pengulangan.

Perhitungan efisiensi isolat protein dengan rumus:

%𝑃𝑟𝑜𝑡𝑒𝑖𝑛 =𝑏𝑒𝑟𝑎𝑡 𝑖𝑠𝑜𝑙𝑎𝑡 𝑝𝑟𝑜𝑡𝑒𝑖𝑛

𝑏𝑒𝑟𝑎𝑡 𝑎𝑤𝑎𝑙× 100%

7.1.1. Efisiensi isolat protein metode phenol/chloroform

Berat sampel awal yang digunakan adalah 15 gram

Berat isolat protein (gram) = 0.025 + 0.026 + 0.056 + 0.018 + 0.024 + 0.019 + 0.28 +

0.018 + 0.02 = 0.486.

Efisiensi isolat protein = 0.486

15 x 100% = 3,24 %

7.1.2. Efisiensi isolat protein metode TCA/Asetone



0.008 + 0.008 = 0.112

Efisiensi isolat protein = 0.112

15 x 100% = 0,75 %

7.1.3. Efisiensi isolat protein metode salting out



0.023 + 0.027 = 0.235

% protein = 0.235

15 x 100% = 1,57 %

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7.2. Perhitungan konsentrasi isolat protein

Konsentrasi isolat protein dihitung dengan menggunakan metode kurva standart metode

Bradford.

Tabel 7. Hasil absorbansi standart BSA (Bovine Serum Albumin)

BSA (ppm) Absorbansi

0 0.0002

10 0.0378

20 0.111

40 0.1878

60 0.2941

80 0.3947

100 0.4475

120 0.4899

140 0.5768

160 0.6306

180 0.7128

200 0.7514

Gambar 10. Kurva standart BSA

7.2.1. Konsentrasi Protein Metode Phenol/chloroform (mg/ml)

Absorbansi ulangan 1 = 0.0486

y.= 0,0049x – 0,0018

0,0486 = 0,0049x – 0,0018

X = 9,9

y = 0.0049x - 0.0018R² = 0.9969

-0.1

0

0.1

0.2

0.3

0.4

0.5

0 20 40 60 80 100

Absobansi

BSA (ppm)

Kurva standart BSA

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Karena 1 kali pengulangan, maka :

Konsentrasi x 10 = 99

Absorbansi ulangan 2 = 0,056

y.= 0,0049x – 0,0018

0,056 = 0,0049x – 0,0018

X = 11,796


Konsentrasi x 10 = 117,959


y.= 0,0049x – 0,0018

0,0565 = 0,0049x – 0,0018

X = 11,898



Rata-rata = 99+117,959+118,979

3 = 112

7.2.2. Konsentrasi Protein Metode TCA/Aseton (mg/ml)


y.= 0,0049x – 0,0018

0,0319 = 0,0049x – 0,0018

X = 6,9




y.= 0,0049x – 0,0018

0,05 = 0,0049x – 0,0018

X = 10,571




y.= 0,0049x – 0,0018

0,0596 = 0,0049x – 0,0018

X = 12,531

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Rata-rata = 69+105,71+125,31

3 = 100

7.2.3. Konsentrasi Protein Metode Salting out (mg/ml)


y.= 0,0049x – 0,0018

0,0278 = 0,0049x – 0,0018

X = 6




y.= 0,0049x – 0,0018

0,0533 = 0,0049x – 0,0018

X = 11,245




y.= 0,0049x – 0,0018

0,0501 = 0,0049x – 0,0018

X = 10,592



Rata-rata = 60+112,45+105,92

3 = 9

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7.3. Hasil Uji Beda Efisiensi isolat protein daun yakon

Tabel 8. Uji Beda Efisiensi isolat protein daun yakon

Dari Tabel 8, diperoleh hasil bahwa terdapat perbedaan antara efisiensi metode isolasi protein pada metode TCA/Acetone dan Salting Out

(0<0,05)

Independent Samples Test

Levene's Test

for Equality of

Variances t-test for Equality of Means

F Sig. t df Sig. (2-tailed) Mean Difference Std. Error Difference

95% Confidence Interval of

the Difference

Lower Upper

Efisiensi Equal variances assumed 1.138 .302 -5.201 16 .000 -.01367 .00263 -.01924 -.00810

Equal variances not

assumed

-5.201 13.986 .000 -.01367 .00263 -.01930 -.00803

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Tabel 9. Uji Beda konsentrasi protein daun yakon

Dari Tabel 9, diperoleh hasil bahwa tidak terdapat perbedaan antara konsentrasi metode TCA/Acetone dengan metode Salting Out. (0,773>0,05)

Independent Samples Test

Levene's Test for

Equality of

Variances t-test for Equality of Means

F Sig. t df Sig. (2-tailed)

Mean

Difference Std. Error Difference

95% Confidence Interval of the

Difference

Lower Upper

Konsentrasi Equal variances assumed .015 .907 .309 4 .773 7.21667 23.33915 -57.58321 72.01655

Equal variances not assumed .309 4.000 .773 7.21667 23.33915 -57.58321 72.01655

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y = -1.0561x + 2.1394R² = 0.9219

0

0.5

1

1.5

2

2.5

0 0.2 0.4 0.6 0.8 1

Log

BM

Rf

Kurva Rf

7.4. Perhitungan Berat Molekul

Tabel 10. Hasil perhitungan Rf dari berat molekul pada marker protein

Berat Molekul (kDa) Panjang pita (cm) Log (BM) Rf

180 0.316 2,2553 0.06389

130 0.382 2,1139 0.07723

100 0.558 2 0.11282

75 0.782 1,8751 0.15811

63 1.148 1,7993 0.23211

48 1.994 1,6812 0.40315

35 2.426 1,5441 0.4905

28 3.286 1,4471 0.66438

17 4.602 1,2304 0.93045

Gambar 11. Kuva Rf pada perhitungan marker protein

Persamaan linear regresi y= -1.0561x+ 2.139

Perhitungan Berat Molekul:

Sumur 2, 3 dan 4 : metode phenol/chloroform

1. Panjang gel : 4,946 cm

Panjang pita = 0.726 cm

x = Rf = 0.726

4,946 = 0,146785

y = 1,984380

BM = antilog y = 97 kDa



x = Rf = 0.726

4,946 = 0,431055

y = 1,6841624


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Panjang pita = 2,29 cm

x = Rf = 2,29

4,946 = 0.463000

y = 1.650425




x = Rf = 0.726

4,946 = 0,515568

y = 1,5949085




x = Rf = 2,644

4,946 = 0,534573

y = 1.574837




x = Rf = 3,02

4,946 = 0.610594

y = 1,494551




x = Rf = 3,306

4,946 = 0,668419

y = 1.433483




x = Rf = 0.726

4,946 = 0,918722

y = 1,169138


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Sumur 5,6 dan 7 : Metode TCA/Aseton

1. Sejajar dengan 180 kDa



x = Rf = 1,684

4,946 = 0,340477

y = 1,7798221




x = Rf = 2,078

4,946 = 0,420138

y = 1,695693


Sumur 8,9 dan 10 : Metode Salting out



x = Rf = 1,244

4,946 = 0,251516

y = 1,873774




x = Rf = 0.726

4,946 = 0,837848767

y = 1,254548


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7.5. Sekuens Asam Amino penyusun protein hasil analisa SDS PAGE dan

Bioinformatika

10 20 30 40 50

MIDRYTHQQL RIGLVSPQQI STWSKKILPN GEIVGEVTKP YTFHYKTNKP

60 70 80 90 100

EKDGLFCERI FGPIKSGICA CGNYRVIGDE KEDPQFCEQC GVEFVDSRIR

110 120 130 140

RYQMGYIKLA YPVMHVWYLK RLPSYIVNLL DKPLNELEDL VYCG

Gambar 12. Sekuens asam amino penyusun RNA polymerase beta subunit (sumber:

www.uniprot.org)

10 20 30 40 50

MFPMVTEFMN YGQQTVRAAR YIGQGFMITL SHANRLPVTI QYPYEKLITS

60 70 80 90 100

ERFRGRIHFE FDKCIACEVC VRVCPIDLPV VDWKLETDIR KKRLLNYSID

110 120 130 140 150

FGICIFCGNC VEYCPTNCLS MTEEYELSTY DRHELNYNQI ALGRLPMSII

160

DDYTIRTILN LPEIKT

Gambar 13. Sekuens asam amino penyusun NAD(P)H-quinone oxidoreductase subunit I,

chloroplastic (sumber: www.uniprot.org)

10 20 30 40 50

MALVNVNSFL AKAESKDLKI DAVRSSELYK DATMLNPSVL NCIKRFETNV

60 70 80 90 100

KVYAQDGQDL CFTDFQLFDK DEIESIRGMS GKYKYVHVGV ILIAIRAMFP

110 120 130 140 150

NYKGKGGRVI VYDGSCIDDD KSQGFIAADE FTFTDDTCYF AIRPSHIFST

160 170 180 190 200

TDAHIADLLR FSIDLDCPKY KDDRELIALD IGVAYRMCNA SRFLDTKGGA

210 220 230 240 250

NNWSHQAIHG CSALEYGPDI ERVLSRPRIP MEVRDRGSNI FERKRIFGKN

260 270 280 290 300

ELRRSRNIEA RRFGRGDEAR FRSGSIRIDR YSKDEFGERY SSDESTQGYS

310 320

IPVGELHRQR KEVEEGTSKS RQP

Gambar 14. Sekuens asam amino penyusun Putative 36.9 kDa movement protein

(sumber: www.uniprot.org)

10 20 30 40 50

YHFQLDDPLI QLVEDYKWIN FFDFRWKLGI DGLSIGPVLL TGFITTLATL

60 70 80 90 100

AAWPVTRDSR LFHFLMLAMY SGQIGSFSSR DLLLFFIMWE LELIPVYLLL

110 120 130 140 150

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SMWGGKKRLY SATKFILYTA GGSIFLLMGV LGVGLYGSNE PTLNFETSVN

160 170 180 190 200

QSYPVALEII FYIGFFIAFA VKLPILPLHT WLPDTHGEAH YSTCMLLAGI

210 220 230 240 250

LLKMGAYGLI RINMELLPHA HSIFSPWLMI VGTIQIIYAA STSPGQRNLK

260 270 280 290 300

KRIAYSSVSH MGFILIGIAS ITDTGLNGAI LQIISHGFIG AALFFLAGTS

310 320 330 340 350

YDRIRLVYLD EMGGVAIPMP KIFTMFSSFS MASLALPGMS GFVAEVIVFL

360 370 380 390 400

GIITSQKYLL MPKIAITFVM AIGMILTPIY LLSMSRQMFY GYKLFNIPNS

410

YVFDSGPREL FVSISIF

Gambar 15. Sekuens asam amino penyusun NADH dehydrogenase subunit 4

(sumber:www.uniprot.org)

10 20 30 40 50

MRMNPTTSSS GVATLDKKNL GRIAQIIGPV LDVAFPPGKM PNIYNALVVK

60 70 80 90 100

GRDTVGQPIN VTCEVQQLLG NNRVRAVAMS ATDGLTRGMD VIDTGAPLSV

110 120 130 140 150

PVGGATLGRI FNVLGEPIDN LGPVDNSTTF PIHRSAPAFI QLDTKLSIFE

160 170 180 190 200

TGIKVVDLLA PYRRGGKIGL FGGAGVGKTV LIMELINNIA KAHGGVSVFG

210 220 230 240 250

GVGERTREGN DLYMEMKESG VINEQNIAES KVALVYGQMN EPPGARMRVG

260 270 280 290 300

LTALTMAEYF RDVNEQDVLL FVDNIFRFVQ AGSEVSALLG RMPSAVGYQP

310 320 330 340 350

TLSTEMGSLQ ERITSTKEGS ITSIQAVYVP ADDLTDPAPA TTFAHLDATT

360 370 380 390 400

VLSRGLAAKG IYPAVDPLDS TSTMLQPRIV GDEHYETAQQ VKQTLQRYKE

410 420 430 440 450

LQDIIAILGL DELSEEDRLT VARARKIERF LSQPFFVAEV FTGSPGKYVG

460 470 480 490

LAETIRGFQL ILSGELDGLP EQAFYLVGNI DEATAKAMNL EMESNLKK

Gambar 16. Sekuens asam amino penyusun ATP synthase subunit beta, chloroplastic


10 20 30 40 50

FYFARPIDKK PTFLRLRGLL EYEIQPWKYR IPIFFTTRSF DTFRNREMST

60 70 80 90 100

GGGSIRQQLA NLDLRMIIDY SLVEWKELEE EEPTGNEWED RKVGRRKDFL

110 120 130 140 150

LRRMELAKHF IRTNIEPKWM VLCLLPVLPP ELRPIYHIDE DKLVTSDINE

160 170 180 190 200

IYRRIIYRNN TLTDLLTTSI ATPEXLIISQ EKLLQEAVDA LLDNGICGQP

210 220 230 240 250

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MRDDHNRIYK SLSDVIEGKE GRVRETLLGK RVDYSGRSVI VVGPSLSLHR

260 270 280 290 300

CGLPREIAIE LFQAFVIRDL IRKHLASNIG VAKSQIRKKK PIVWEILQEI

310 320 330 340 350

LDDHPVLLNR APTLHRLGIQ AFLPVLVEGR AICLHPLVCK GFNADFDGDQ

360 370 380 390 400

MAVHVPLSLE AQAEARLLMF SHMNLLSPTI GDPISAPTQD MLSGLYVLTS

410 420 430 440 450

GNRRGICVNR YNPCNRRNYQ NEDNNYKYTK KKEPFFCNAY DAIGAYRQKR

460 470 480 490 500

INLGSPLWLR WRLDQRVIAA REAPIEIHYE SLGTYYEIYG HYLIVRSIKK

510 520 530 540

EILYIYIRTT LGHISLYREI EEAIQGFWQG CCNSMLPAGI RVSPG

Gambar 17. Sekuens asam amino penyusun DNA-directed RNA polymerase subunit


10 20 30 40 50

MEQTYQYAWI IPFLPLPVPM LIGLGLLLFP TATKSLRRMW AFQSVLLLSI

60 70 80 90 100

VMIFSMNLSI QQINSSSVYQ YVWSWIINND FSLEFGYLID PLTSIMSILI

110 120 130 140 150

TTVGIMVLIY SDNYMSHDHG YLRFFAYMSF FSTSMLGLVT SSNLIQIYIF

160 170 180 190 200

WELVGMCSYL LIGFWFTRPV AAKACQKAFV TNRVGDFGLL LGILGFYWIT

210 220 230 240 250

GSFEFRDLFQ IFNNLISNNE VNFVFVTLCA VLLFAGAIAK SAQFPLHVWL

260 270 280 290 300

PDAMEGPTPI SALIHAATMV AAGIFLVARL MPLFIVIPHI MNFISLIGII

310 320 330 340 350

TVFFGATLAL AQKDIKRGLA YSTMSQLGYM MLALGMGSYR SALFHLITHA

360 370 380 390 400

YSKALLFLGS GSVIHSMETL VGYCPKKSQN MVLMGGLTKH VPITKNSFLL

410 420 430 440 450

GTLSLCGIPP LACFWSKDEI LNDSWLYSPI FAIIAWSTAG LTAFYMCRIY

460 470 480 490 500

LLTFEGHLNV HFQNYSGKRN TPLYSISLWG KEGSKISNKN FRLVTLLKMK

510 520 530 540 550

KNGRPSFFSN KVYKMDENVR NLIQPFLSIP NFGNTKTYLY PYESDNTMLF

560 570 580 590 600

PILILILFTL FVGFLGIPFN QDVDILSKWL TPSINLLHKN SNNSIDWYEF

610 620 630 640 650

CKDAVFSVSI ASFGIFISFF LYKPVYSSFQ NLDLINSVVK MGPKRIFSDK

660 670 680 690 700

IKNAIYDWSY NRGYIDAFYG TFLTVGVRRL AEFTHFFDRR IIDGIPNGVG

710 720 730 740

FMSFFVAEVI KSVGGGRISS YLFFYFSYVS IFLLIYYFIK SL

Gambar 18. Sekuens asam amino penyusun NAD(P)H-quinone oxidoreductase

subunit 5, chloroplastic (sumber:www.uniprot.org)

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10 20 30 40 50

KDLQKKFFQQ RCELGGIGRR NMNRRLNLDI PQNNTFLLPR DILAAADRLI

60 70 80 90 100

RIKFGMGTLD DMNHLQNKRI RSVADLLQEQ FGLALVRLEN MARGNIYAAL

110 120 130 140 150

KHNWTPTPQN LVNSTPLTDT YKVFFRLHPL SQVLDRTNPL TQIVHGRKLS

160 170 180 190 200

YLGPGGLTAR TATFPIRDIH PSHYGRICPI DTSEGINVGL IGSLAIHARI

210 220 230 240 250

GRWGSLESPF YKISERSKGA QMLYLSPGRD EYYMVAAGNS LALNQGIQEE

260 270 280 290 300

QVVPARYRQE FLTIAWEQVH LRSIFAFQYF SIGASLIPFI EHNDANRALM

310 320 330 340 350

SSNMQRQAVP LSQSEKCIVG TGLEGQAALD SGALAIAEHE GKIFYTDTDK

360 370 380 390 400

ILLSGNGDTL RIPLVMYQRS NKNTCMHQKP QVRRGKCIKK GQILAYGAAT

410 420 430 440 450

VGGELALGKN VLVAYMPWEG YNFEDAVLIS ERLVYEDIYT SFHIRKYEIQ

460 470 480 490 500

INQGPERVTN EIPHLEVHLL RNLDKNGIVM LGSWVETGDI LVGKLTPQMV

510 520 530 540 550

KESSYAPEDR LLRTILGMRV YTSKETCLKL PIGGRGRVID VRWVQSSKTD

560 570 580 590 600

ETEKTESIRV YILQKREIKV GDKVAGRHGN KGIISKILPR QDMPYLQDGR

610 620 630 640 650

PVDMVFNPLG VPSRMNVGQI FESSLGLAGD LLDRHYRIAP FDERYEQEAS

660 670 680 690 700

RKLVFSELYE ASKQTANPWI FEPESPGKSR IFDGRTGDPF EQSVIIGKPY

710 720 730 740 750

ILKLIHQVDD KIHGRSSGRY SRLTQQPLKG RAKKGGQRVG EMEVWALEGF

760 770 780 790 800

GVAYILQEML TYKSDHIRAR QEVLGTIIFG GRIPTPEDAP ESFRLFVREL

810 820

RSLALELNHF LVSEKTFQLN RKEA

Gambar 19. Sekuens asam amino penyusun DNA-directed RNA polymerase subunit

beta (sumber: www.uniprot.org)

7. LAMPIRAN 7.1. Perhitungan efisiensi isolat proteinrepository.unika.ac.id/14666/8/12.70.0154...

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