رايط الشرح علي اليوتيوب https://youtu.be/WLqHg3ZUT9s

1. PROTEIN ELECTROPHORESIS
BY
dr: Mohammed Bahgat Mohammed
Sofyan
Assistant lecturer of medical biochemistry,
Faculty of medicine, Al-Azhar university
(Assiut branch)
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2. 2

3. 3

4. ‫صورة‬ ‫كل‬ ‫على‬ ‫اضغط‬
‫الشاش‬ ‫عرض‬ ‫وضع‬ ‫في‬
‫ة‬
‫قنواتنا‬ ‫على‬ ‫وادعمنا‬
‫اإلنترنت‬ ‫على‬

5. ‫على‬ ‫ملفي‬ ‫في‬ ‫هنا‬ ‫أرفعها‬ ‫التي‬ ‫شرائحي‬ ‫ومذاكرة‬ ‫بالتعلم‬ ‫وأسعد‬ ‫وأرحب‬ ‫أسمح‬
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‫مانع‬
‫أيضا‬
‫من‬
‫نسخ‬
‫شريحة‬
‫او‬
‫اثنين‬
‫عند‬
‫الضرورة‬
،
‫وال‬
‫مانع‬
‫من‬
‫شرح‬
‫البورب‬
‫وينت‬
‫الخاصة‬
‫بي‬
‫للغير‬
‫بشرط‬
‫عدم‬
‫إزالة‬
‫اسمي‬
‫من‬
‫البوربوينت‬
،
‫فال‬
‫أسمح‬
‫أبدا‬
‫بإزال‬
‫ة‬
‫اسمي‬
‫من‬
‫على‬
‫الباوربوينت‬
‫ووضع‬
‫اسمك‬
‫بدال‬
‫منه‬
‫لتصبح‬
‫وكأنك‬
‫من‬
‫صممتها‬
‫فهذه‬
‫سرقة‬
‫ال‬
‫أسمح‬
‫بها‬
‫وتضييع‬
‫لحق‬
‫من‬
‫تعب‬
‫في‬
‫عملها‬
.
‫وفقكم‬
‫هللا‬
‫وإياي‬
‫للتعلم‬
‫ونفع‬
‫اآلخرين‬
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There is also no objection to copying one or two slides when
necessary, and there is no objection to explaining my PowerPoint to
others on the condition that my name is not removed from the
PowerPoint. I never allow my name to be removed from PowerPoint
and to replace it with yours, Make it look like you designed it. This is
theft that I do not allow and a waste of the right of those who are
tired in this work. May God bless you and me for learning and
benefiting others
5

6. Definitions
Electrophoresis is the migration of
charged particles in an electrical
field.
Or it is a method used to separate
charged particles from one another,
based on differences in their migration
speed i.e. The charged particles move in
an electric field towards the oppositely
charged electrode.
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7. RACING
7

8. 8

9. importance
By electrophoresis, a mixture of
amino acids, proteins,
lipoproteins, isoenzyms, CSF
proteins, DNA fragments, Hb
variant, etc…. can be separated
by using electric current.
9

10. protein buffer
10

11. PRINCIPLE
Proteins are amphoteric compounds and are therefore
either positively or negatively charged because they
contain both acidic and basic residues.
In an alkaline buffer (pH 8.5-9), serum proteins gain
negative charges and migrate to the positive electrode
(anode).
On a support media, proteins are separated according
to their shape, size and net surface charge , they
migrate to anode.
11

12. Ph of solution
• Proteins have amphoteric properties i.e. it can carry both positive and negative
charge
• At specific pH of solution called isoelectric point (pI) of this protein , is the pH
at which a molecule carries no net electrical charge, proteins carry no net
charge.
• Below this PH ( acidic pH ) proteins carry positive charge.
• Above this PH ( alkaline pH ) proteins carry negative charge.
12

14. instrumentation
• Apparatus: contains the electrodes, buffer reservoirs, area for the support
medium & a cover to minimize evaporation and protect system.
• Power supply: constant-current power supply.
• Buffer: carry applied current, fix PH, determine electrical charge & extent
of ionization.
• Support media: as cellulose acetate, Agarose gel & Polyacrylamide gel,….
• Agarose gel is used in most immunoelectrophoretic techniques because of
the large pore size allowing Ag and Ab to migrate through the gel.
14

15. 15

16. Factors affecting
electrophoresis
A. Sample:
Charge , Size & Shape.
B. The Supporting medium:
Adsorption , Molecular Sieving & Electro-osmosis
(Electro-Endosmosis).
C. Buffer:
Composition , Concentration (Ionic strength) & PH
(8.6).
D. Electric Field:
Voltage , Current , Resistance & Heat
16

17. Types
A. Zone Electrophoresis: migration of charged molecules as zone.
They carried on constant pH, they classified according supporting
media into:
1. Agarose gel electrophoresis (AGE): For analysis of serum protein,
nucleic acids,LP, hemoglobin variants, CK isoenzymes, LDH
isoenzyme
2.Cellulose acetate electrophoresis (CAE): usually serum proteins.
3. Polyacrylamide gel electrophoresis (PAGE): For analysis of serum
protein, nucleic acids in serum esp. genetic variants and
isoenzymes. It is thermostable, Prepared in a variety of pore sizes. Better
resolution and fractionation for serum proteins (up to 30 fractions).
4. Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis (SDS-
PAGE): separation is based on molecular size only ( detecting MW),by
adding sodium dodecyle sulfate (SDS) that make protein negative.
17

18. AGROSE GEL ELECTROPHORESIS 18

19. Types
C. Isoelectris focusing (IEF):
Proteins move to zones where PH medium = pI,
where the charge = 0 and migration is stopped , so
we can seprate proteins by this methods as each
protein has its pI.
Proteins that differ in their pI by only a 0.02 pH
unit are seprated by IEP, SO pI of protein is
confined in a narrow PH range leading to sharp
protein zones.
The PH gradient is created with LMW carrier
ampholytes.
D. Two-Dimentional (2D) Electrophoresis:
IEF+SDS (PAGE)
19

20. ISOELECTRIC FOCUSING 20

21. TWO DIMENTIAL ELECTROPHORESIS
21

22. Types
E. Capillary Electrophoresis (CE):
Using a small-bore, Fused-silica capillary tube 20–200 cm
long.
Can be used with high voltages (25-30 KV) enhancing
separation efficacy and reducing separation time.
The ease of automation.
F. Blotting techniques:
Southern Blotting : For separation of DNA or its fragments
on by AGE.
Northern Blotting: For separation of RNAs or their
fragments.
Western Blotting : To identify one or more proteins in a
complex mixture using SDS-PAGE and antibody.
22

23. Principle of the Procedure
1. Sample (e.g. serum containing a mixture of proteins) is applied to a strip of filter paper or
cellulose acetate. Then both edges of the strip is dipped in alkaline buffer solution.
2. Because proteins are amphoteric, they will carry negative charges in alkaline medium.
3. When the current passes, proteins will migrate towards positive electrode (anode). The rate of
migration depends on:
a) The amount of charges carried by each protein.
b) The molecular weight and size of proteins.
4. By this method, serum proteins can be separated into several bands, each band represents
special type of protein. These types are: albumin, globulins[a (a1,a2),b and g globulins].
5. The density of each band is directly proportional to its serum concentration. So albumin shows
the densest band.
6. After electrophoretic sepration the plate is stained to locate and visualize the individual protein
zone.
7. Direct densitometer is used quantify the individual zone.
23

24. Serum proteins in electrophoresis
24

25. DENSITOMETRY 25

27. SUMMARY
‫ال‬ ‫جهاز‬ ‫طريق‬ ‫عن‬ ‫الدم‬ ‫في‬ ‫اللي‬ ‫البروتينات‬ ‫هنفصل‬ ‫معانا‬ ‫اللي‬ ‫الطريقة‬ ‫في‬
protein electrophoresis
‫في‬ ‫البروتينات‬ ‫هنجري‬
electric field
‫ال‬ ‫حسب‬ ‫علي‬ ‫هيجروا‬
Molecular weight and charge
‫الي‬ ‫ويتفصلوا‬
5
bands
‫كالتالي‬
:
1_ albumin
2_ Alfa 1 globulin
3_ Alfa 2 globulin
4_ B globulin
5_ gamma globulin
‫للعينات‬ ‫هنعمل‬
loading on the gel
‫عليه‬ ‫اللي‬
buffer
‫ب‬ ‫الكهرباء‬ ‫ونوصل‬
Specific current and voltage
‫ال‬ ‫على‬ ‫تجري‬ ‫العينات‬ ‫تقوم‬
gel
‫ال‬ ‫حسب‬ ‫علي‬
Molecular weight and charge
‫حوالي‬ ‫بعد‬
3/4
‫في‬ ‫وتوضع‬ ‫العينة‬ ‫تنشف‬ ‫ساعة‬
stain
‫ال‬ ‫يصبغ‬
band
‫بال‬ ‫أشوفها‬ ‫علشان‬
direct densitometer
27

28. 28

29. 29

30. 30

31. 31

32. 32

33. 33

34. 34

35. 35

36. 36

37. 37

38. Polyclonal
hypergammaglobulinaemia
¨ Chronic liver disease
¨ Chronic infections
¨ Inflammatory disease of bowel
¨ Autoimmune disorder
¨ Granulomas
•
‫ونلخصها‬
‫بال‬
• Chronic infection and inflammation
38

39. Causes of Monoclonal
hypergammaglobulinaemia
‫الجل‬ ‫علي‬ ‫بتعمل‬ ‫دي‬
sharp localized band as :
• Benign
• Idiopathic
• Diabetes mellitus
• Chronic infections
39

40. Causes of Monoclonal
hypergammaglobulinaemia
• Cirrhosis
• Connective tissue disorders
• Malignant
• Multiple myeloma
• Macroglobulinaemia
‫بال‬ ‫ونلخصها‬
B cell cancer
40

41. Monoclonal Gammopathies
41

42. REFERENCES :
• _ https://labtestsonline.org
• _ https://www.medscape.com
• _ https://www.wikipedia.org
• _ https ps://www.labcorp.com
• _ https://www.uptodate.com
• _ https://www.ncbi.nlm.nih.gov Home - PubMed – NCBI
• _TIETZ TEXTBOOK OF CLINICAL CHEMISTRY AND MOLECULAR DIAGNOSTICS, SIXTH EDITION 2018.
• _Essential of clinical pathology book; 1st edition; Shirish M Kawthalkar; 2010.
• _Essential of biochemistry book ;1st edition; 2012.
• _ ABC of clinical hematology ; 4th edition ; Drew Provan; 2018.
• _ Harper's illustrated biochemistry 30th edition 2015.
• _ Lippincott's illustrated review of biochemistry sixth edition 2014.
• _ Lecturea Notes Clinical Biochemistry, 9th Edition Walker, Simon, 2103.
• _Some audios and videos from Well-known, trusted professors who study from accredited books.
42

43. ‫اليوتيوب‬ ‫علي‬ ‫البحث‬ ‫أو‬ ،‫اليوتيوب‬ ‫علي‬ ‫قناتي‬ ‫دي‬
‫د‬ ‫باسم‬
.
‫سفيان‬ ‫بهجت‬ ‫محمد‬
،
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‫حتى‬ ‫بالقناة‬ ‫ا‬
‫هللا‬ ‫شاء‬ ‫إن‬ ‫نستمر‬
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‫ب‬ ‫الفيس‬ ‫في‬ ‫البحث‬ ‫أو‬ ،‫انضمامكم‬ ‫يشرفنا‬ ‫الفيسبوك‬ ‫علي‬ ‫بتاعتي‬ ‫الصفحة‬ ‫دي‬
DMBMS2018
،
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‫هللا‬ ‫شاء‬ ‫إن‬ ‫نستمر‬
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