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Protein purification مه أداا
Page 2 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 .
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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
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. 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
Protein purification مه أداا
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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
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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
Protein purification مه أداا
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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
Protein purification مه أداا
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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.
Protein purification مه أداا
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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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الفرق الطب األكادم
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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الفرق الطب األكادم
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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الفرق الطب األكادم
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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الفرق الطب األكادم
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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الفرق الطب األكادم
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
Protein purification مه أداا
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الفرق الطب األكادم
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
Protein purification مه أداا
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الفرق الطب األكادم
purification كل منها تذهب لل fragments نتج trypsinال
Protein purification مه أداا
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الفرق الطب األكادم
% 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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الفرق الطب األكادم
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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الفرق الطب األكادم
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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الفرق الطب األكادم
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.”