Protein purification اٌإدأ هم - كلية الطبProtein purification ا إدأ هم 2Page...

Protein purification مه أداا

الفرق الطب األكادم



Protein Purification & Characterizing Techniques

1. How Do We Extract Pure Proteins from Cells?

Disruption of cells is the first step in protein purification. The various parts

of cells can be separated by centrifugation. This is a useful step because

proteins tend to occur in given organelles. High salt concentrations will

precipitate groups of proteins, which are then further separated by

chromatography and electrophoresis.

قبلها الزم نعمل centrifugationلو عندك نوع من انواع اللحم و بدك تعرف شو نوع البروتن الل فه بنعمل

precipitation اوsalting out نحطammonia sulfate بنعملbuffer .

لصبح crushingو بنعمله buffer الل بدنا ااه نضعه مع لو بدنا نعرف اي بروتن موجود باالش

homogeneous solution بعد ال ,centrifugation رح ترسب بحث نفصل البروتنات حسب السرعة و

و العنات chromatographyو بنعمل supernatantبنوخد ال centrifugationحسب النوع بعد ما خلص ال

و بنشوف اش محتواتها . electrophoresisع بنعمللها الل بتطل

2. What Is Column Chromatography?

Two of the most important methods for separating amino acids,

peptides, and proteins are chromatography and electrophoresis. The

various forms of chromatography rely on differences in charge,

polarity, or size of the molecules to be separated, depending on the

application.

Column in different sizes و بكون عندناstationary phase و العنةmobile phase, و بعتمد ال

separation حسب الcharge, polarity and size of molecule.

3. What Is Electrophoresis?

In electrophoresis, differences in charge and in size are the criteria

for separation. The sieving action of gel slabs is used in conjunction

with the charge on proteins to achieve separation. The

electrophoretic mobilities of proteins can be used to estimate their

molecular weights.

molecularانه نعرف ال electrophoresisعن الهدف من ال charge and sizeبعتمد ع ال

weight الخاص بالبروتن الل هوknown protein .



4. How Do We Determine the Primary Structure of a Protein?

Determination of the N-terminal and C-terminal amino acids of

proteins depends on the use of these separation methods after the

ends of the molecule have been chemically labeled. Selective

cleavage of the protein into peptides by enzymatic or chemical

hydrolysis produces fragments of manageable size for sequencing

PROTEINS & PEPTIDES MUST BE

PURIFIED PRIOR TO ANALYSIS Highly purified protein is essential for determination of its amino acid sequence. Cells contain thousands of different proteins, each in widely varying amounts. The isolation of a specific protein in quantities sufficient for analysis presents a challenge that may require multiple successive purification techniques.

و بنحصل على الجدول buffer and salting out different concentrationنعمل

هذا الجدول مهم *********

النه المحلول percent recovery =100نجد انه ال buffer مع crushing الخلط البدائ الل عملتله .1

كامل



. clear solutionبنوخد الشوائب و بنحصل على precipitation ال انخفض الن ب volumeال .2

***decrease quantity –decrease total protein –decrease percent recovery بس الل

specific activity =total activity/total proteinبزد

highest او عن smallest amount of proteinعن highly purified عن لما كون االنزم

amount of specific activity .

specific activity for enzyme or protein بنبحث عن ال purification techniqueال ف

الل specific activityل ال و فها ك one dropالعبوة فها mg ولس بال µgاالنزم بنباعوا بال

بنبحث عنها .

Dialysis.

Protein molecules (red) are retained within the dialysis bag, whereas small molecules (blue) diffuse

Dialysis

.separate protein according to their sizeو تعن purificationمن خطوات ال

small molecule will passعن ال sulfate bags semipermeable membraneبكون ف عندنا

و بتكون مثل البكرة و نضع فه البروتن الل بدي اعمله large molecule will trapped بنما ال

separation و فهbuffer حواله و بتعملهstraining و بضل تحرك الى ساعات و بعدن بنوخد الل

large molecule ال solutionالى sulfate bag برا ال الل رح طلع, small moleculeبرا ال

. trapped insideرح ضلوا

Centrifugation الهdifferent speeds, sizes and types ومن انواعهultra-centrifugation

(one million gravity), small and refrigerated.

Separation of cells عتمد على انهwhere the precipitation will take place



Nuclei and broken cell 600رح ترسبوا على سرعةG 10ف min و بنحصل علىsupernatant1

,mitochondriaو ستترسب ال centrifugationو دائما بنوخذه و نعمله electrophoresis رح نستعمل

lysosomes and microbodies و بعدن بنوخذ الsupernatant2 100000وبنكمل ع سرعةG لمدة

… ribosome, Golgi apparatus, ER, plasma membraneساعة و ترسب

ساعة لرسب اصغر شء بالخلة 22ل centrifugationمكن نخل ال

و بتشوف electron microscope و بتشوفه بال supernatant and precipitationبنوخذ ال

مكوناتها.

Gel-Filtration Chromatography



2 phases:

stationary phase -powder we will put it in column1-

Mobile phase-sample2- العنة دائما بنحط معها صبغات لنمز الseparation stages ي مرحلة أل و

وصلت.

لصبح محلول ionized waterعبارة عن باودر بنغسل اكثر من مرة ب columnف ال powderال

homogenizes بعدن بنضفه للcolumn و بنزل الزادة من الماء و لما صبحcompletely

compact عن ما فهcracks و صبح قطعة واحدةhomogenies بعد ذلك بنضف علهbuffer و

وبنوخذهم و بنكمل لالخر. fraction collectionبنعمل

لحتى نشف. buffer continuousرح ضل عله columnو ال

Agarose- sugar

columnالل رح نحطها بال substancesواحد من انواع ال

Gel filtration-size exclusion chromatography.

عتمد على الحجم separationال



Small molecule (beads) will enter into the beads of column, large molecule will not

enter into beads.

Fraction collection ف منهautomatic 3عن تحددml ف كلtube الحجم الل بدنا ااه وهو لحاله

tube and so onو بنتقل لثان switchبعمل

smallرح طلع ال bufferوبعدن لما نضف large moleculeالل رح نفصل باالول هو ال

molecule

Affinity Chromatography

Column with substance S (supporting material)

The column has substance and already we put in it another substance

P1 رح تحدوا معs ,الموجودةp2 and p3 ما رح تحدوا



عن substanceفصل ال buffer high concentrationزد طلعوا بن p2 and p3كد انه بعد ما اتأ

p1 الموجودة ف الcolumn .

Affinity Chromatography Affinity chromatography of concanavalin A (shown in yellow)

on a solid

support containing covalently attached glucose residues (G).

The plant protein concanavalin A can be purified by passing a crude extract through a column of beads containing covalently attached glucose residues. Concanavalin A binds to such a column because it has affinity for glucose, whereas most other proteins do not. The bound concanavalin A can then be released from the column by adding a concentrated solution of glucose.

Ion-Exchange Chromatography. This technique separates proteins mainly according to their

net charge.



column charged positively or negatively

Positive بتحد معnegative و بطلعpositive beads

column anion and cationلتال ف نوعن من ال او ب

structureالزم نعرف االسماء بدون ال *********


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الزم مر بكل الخطوات مرة و مرتن وثالث لحتى نحصل على protein purificationلما بتبحث عن ال ****

protein purified نحصل على الprotein with charge

techniques, identificationلحتى نحصل على االنزمات الغالة جدا الت نستخدمها ف ال purificationنعمل

for tumor ,slides for histology and pathology لنتعرف على اشاء محددة.


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Chromatographic Separations

Partition molecules between two phases, one mobile and the other

stationary.

For separation of amino acids or sugars, the stationary phase, or

matrix, may be a sheet of filter paper (paper chromatography) or a thin

layer of cellulose, silica, or alumina (thin-layer chromatography

وجود تنك كبر فه ال بتصر من خالل chatomatographic زمان كان ال

solution و بعدن بنحط العنات على الpaper sheat و بتعلق داخل التنك و

اخد العنات و بمش فها buffer بعدت تلقائا بصر ال

و التوضح ......لالطالع

https://www.youtube.com/watch?v=yosecfE98Ok

Electrophoresis

Is the process of separating compounds on the basis of their

electric charge & size

electrophoresis of amino acids can be carried out using paper,

starch, agar, certain plastics, and cellulose acetate as solid supports

in paper electrophoresis, a paper strip saturated with an aqueous

buffer of predetermined pH serves as a bridge between two

electrode vessels

a sample of amino acids is applied as a spot (the origin) on the

solid support strip

an electric potential is applied to the electrode vessels and amino

acids migrate toward the electrode with charge opposite their own

(positive ترزح نم negayive ال negativeترزح نم positive)ذؼى ان ال

molecules with a high charge density move faster than those with a

low charge density

molecules at their isoelectric point remain at the origin

after separation is complete, the strip is dried and developed to

make the separated amino acids visible




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كن ال wellsذضغ انؼىاخ ف انفرذاخ ان ف انجم ذسم

Electrophoresis مصل تثطارح ذصم الseparation

horizontal Electrophoresisسم ذ

نالطالع انرضخ النح انؼمم

https://www.youtube.com/watch?v=vtxb6Tr8Y3s

Polyacrylamide Gel Electrophoresis

ventricle Electrophoresisهذا هو ال

ف الوعاء pufferكون الجل بن لوحن من الزجاج و من ثم وضع ال

اللة العمل لالطالع و التوضح

https://www.youtube.com/watch?v=eaETFKXtNRA

Direction of movement from the top to the bottom in different

levels






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ان dryer رم اخذ انجم ان ماكىح اخز ذسم ال separation تؼذ اوراء ال

نهجم مه ثم وذصم ػه staining مه ثم رم ػمم solutionترىشف انجم مه ال

انؼىاخ انمفصنح كما ف انشكم

Electrophoretic Analysis of a Protein Purification.

ال ىا standardجة ان ذ ػه electrophoreticكم

homogenate ال standard انثاق م ال un-known انؼىاخ

انمذرهفح

غر proteinsكون لدك بالبداة بمرور العملات المختلفة على العنة فأنه

معروفة بالمئات و بعدن شوي شوي بتصر اخف و اقل الى ان تصل الى

affinity chromatography نهاة الطرق ال ه

mobilityله اعلى molecular weight االقل


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2 Dimensional gel electrophoresis

ترصم كزتا تذصم tube كم ػىح ترىذط نذال ب tubesتكن ػىا

separation( electrophoresis) تؼذه تىاخذ انجم تىطهؼ مه ال tube تىذط

فكم ادذ مىا رح ىفصم مزج اخز الوا another separation techniqueػه

انر different molecular size كاود ذذ ػه كثز تزذه , فثصز ػىا

SDS Polyacrylamide slab ذسم ب

bandوذذد مكواخ كم separationتؼذ اوراء ال

molecular weights of sample proteins


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simple proteinالزم تحط وحدة من هدول ال electrophoresis لعمل ال

molecular weight to the unknown عشان نعرف ال

بتعط معلومة عن البروتن ال بتستخدمه ف molecular weight هاي ال

العملة و من ثم تستطع تحدد الخصاىص العامة

How Do We Determine the Primary Structure of a

Protein

?

فممكن خضع للتجزئة بواسطة polypeptide linkage البروتن بما انه من

عمل على ) االنزمات ( و كل منها chymotrypsin و trypsin االنزمات مثل

L-Terminal , N-Terminalامنو اسد محدد اما من ال

certain amino acid either from L or N terminal كل انزم عمل على


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purification كل منها تذهب لل fragments نتج trypsinال


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% 07بوجود C-Terminal peptideتحصل على ال chemical cleavageال

HCOOHمن ال

labeling to amino و بعمل polypeptideبنحطه على ال reagentهاد ال

acids و بكل مرة بشل واحد

و رجعنا حطنا ثان مرة ف alanineال residueحطنا اول مرة ف شال اول

circleوكذلك على شكل glycineال residue شال ثان


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Specific cleavage of polypeptides

Cleavage side و انواع ال القطع موقع reagent .....Chemical &

enzymatic

بالتفصل الدكتور قرأ الجدول كامل و حكى ال تخافوا هاد رح رجع نعاد بالسستمات

!!☻.. فما بعرف اذا حفظ او ال


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compoundبنقدر نحدد ال resultمن خالل ال

overallكم مزكة تطهغ ان تؼذي كما ف انصرج ف االخز تطهغ ػىا ال

sequence ان تجمغ االشاء انمشرزكح تىا

ف مخرثزاخ مرطرج ظفرا ذؼطا انثزذه ذؼطك automat كم اد انك تىؼمم

DNA انمكوح نهؼىح ذاػرك ا اوك ذؼط ال amino acid sequence ال

base sequence to the DNA or RNAذطهة

؟! دسة سؤال تىد او كف ك

تأو ادىا تذوا وؼزف ال composition structure to the protein مه خالل

ذثؼ , ؼى ادىا ػىا انثزذه تذوا وؼزف اال composition sequenceمؼزفح ال

amino acid sequence مه ش تركن ف تاالل تىؼمم cutting تؼذه تىسع

تؼذه تىشف اش انمشرزك تىم ) س identification تىؼمههم cutting ال

اخز صرج (


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اضافح جشء مىا مه قثم ذم ذؼذها amino acids مالدظح مه مذاضزج ال

-انذكرر :

Essential amino acids :

Is 10 in children & 8 in adult , there is histidine and arginine

essential in children and not essential in adult

The body cannot synthesis it

The body need them , they must be taken by food

They are of animal origin

their deficiency will lead to disease

Example for protein contain all essential amino acids :-

1. Milk protein

2. Animal meat

3. Eggs

Non-Essential amino acids :

The body can synthesis them

They are not need for the body

They are of plant origin

There deficiency not lead to disease

Example :-

1. Plant protein in Legumes البقولات

}} معناف حال وجود اي خطأ او معلومة ناقصة رجى التواصل {{

ولكم منا جزل الشكر

انتم بخر م وكل عا وبالتوفق

“SucceSS meanS doing the beSt we can with

we have. Success is the doing, not the what

getting; in the trying, not the triumph.

Success is a personal standard, reaching

for the highest that is in us, becoming all

that we can be.”

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Transcript of Protein purification اٌإدأ هم - كلية الطبProtein purification ا إدأ هم 2Page...