Protein electrophoresis is a method used to separate proteins in a blood sample using an electric current. In the procedure, the serum sample is loaded onto a gel support medium like cellulose acetate. An electric field is applied using a buffer solution, causing the proteins to migrate based on their molecular weight and charge. The proteins separate into bands representing different protein types, such as albumin, alpha 1 globulin, alpha 2 globulin, beta globulin, and gamma globulin. After the bands form, the gel is stained to visualize the proteins, then a densitometer can quantify the amount of each band. This technique allows analysis of a protein mixture and detection of abnormalities.

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

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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‫من‬
‫نسخ‬
‫شريحة‬
‫او‬
‫اثنين‬
‫عند‬
‫الضرورة‬
،
‫وال‬
‫مانع‬
‫من‬
‫شرح‬
‫البورب‬
‫وينت‬
‫الخاصة‬
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‫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
I allow, welcome, and be happy to learn and study my slides that I
upload here in my profile on Slide share website
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.
6

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. 10

11. protein buffer
11

12. 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 6.5-9), 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.
12

13. 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 and does not move in electrical field.
• Below this PH ( acidic pH ) proteins carry positive charge.
• Above this PH ( alkaline pH ) proteins carry negative charge.
13

15. 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.
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,…..
Both depends on charge
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.
Both depends on size
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.
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. 23

24. 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 or other, 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[Alfa (a1,a2),Beta and gamma 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.
24

25. Serum proteins in electrophoresis
25

26. DENSITOMETRY 26

27. STAIN 27

28. DETECION AND QUANTIFICATION 28

29. SUMMARY
‫ال‬ ‫جهاز‬ ‫طريق‬ ‫عن‬ ‫الدم‬ ‫في‬ ‫اللي‬ ‫البروتينات‬ ‫هنفصل‬ ‫معانا‬ ‫اللي‬ ‫الطريقة‬ ‫في‬
protein electrophoresis
‫للعينات‬ ‫هنعمل‬
loading on the gel
‫في‬ ‫البروتينات‬ ‫هنجري‬
electric field
‫عليه‬ ‫اللي‬
buffer
‫ال‬ ‫حسب‬ ‫علي‬ ‫هيجروا‬
Molecular weight and charge
‫ب‬ ‫الكهرباء‬ ‫ونوصل‬
Specific current and voltage
‫ال‬ ‫على‬ ‫تجري‬ ‫العينات‬ ‫تقوم‬
gel
‫ال‬ ‫حسب‬ ‫علي‬
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
‫حوالي‬ ‫بعد‬
3/4
‫في‬ ‫وتوضع‬ ‫العينة‬ ‫تنشف‬ ‫ساعة‬
stain
‫ال‬ ‫يصبغ‬
band
‫بال‬ ‫أشوفها‬ ‫علشان‬
direct densitometer
29

30. PROTEIN ELECTROPHORESIS
30

31. UYJ,[[‘0cJJ
JUJollectio
n‫يءؤريبرب‬
‫ريبر‬
Speicment ➡ fresh Serum
No special prepration is required
31

32. UUUUUUU
X XZXSASZS
Avoid plasma and hemolyzed specimen.
A grossly widened albumin zone may be
due to certain medication.
Sample can be stored at room temperature
for 4 days, 4°C for 2 weeks, or at -20°C for 6
month.
32

33. 33

34. Technical
consiHHHH
HHHdration
Supporting media : may be cellulose
acetate , agrose gel or
SDS(PAGE),…. usually cellulose
acetate.
Buffer at pH 6.8 : may be
Barbital buffer or Tris-barbital
with calcium lactate.
Power supply :constant voltage
about 90-100 V within 30 minutes to
decrease the heat and resistance.
Finally, Periciptate proteins and stain
it by Ponceau S, Amido Black or
Coomassie Brilliant,… and detection
by direct densitometer.
34

35. Serum proteins in electrophoresis
35

37. DENSITOMETRY 37

38. NORMAL
VALUES
38

39. 39

40. 40

41. hypoalbuminaemia
albumin ➡ represent about 60% of total plasma proteins
‫ال‬ ‫حاالت‬ ‫في‬ ‫اال‬ ‫يزيد‬ ‫ال‬ ‫ده‬ ‫طبعا‬
dehydration
‫حاالت‬ ‫في‬ ‫وبيقل‬
CAUSES OF HYPOALBUMINEMIA :
1_ LCF ➡liver can't synthesise albumin
2_ RF and nephrotic S ➡ protein loosing in urine Also DM, HT, and
another causes of protenuria.
3_ protein loosing entropathy. ➡ loose protein in stool.
4_ Malnutrition and malabsorbtion.
5_ Acute and chronic infections ( most common cause)
6_ Hemodilutions ‫وغيره‬ ‫المحاليل‬ ‫في‬ ‫زي‬
7_ genetic diseases ➡ rare disease. may reach 0.05 gm /dl.
8_ ascites may be result and cause of hypoalbuminemia!!!!
41

42. 42

43. Alfa 1 globulin
Alfa 1 globulin ➡ in this area there are :
a_ Alfa 1 antitrypsin
‫واحد‬ ‫أهم‬ ‫ده‬
it’s positive acute phase reactant and
decreased in Alfa 1 antitrypsin deficiency
b _ Alfa 1 glycoprotein
c _ Alfa 1 fetoprotein
‫معظم‬
‫هذه‬
‫المجموعة‬
LMW
‫لذلك‬
‫يقلوا‬
‫في‬
‫حاالت‬
Nephrotic Syndrome
43

44. 44

45. Alfa 2 globulin
Alfa 2 globulin ➡
a_ ceruoplasmin
b_ Alfa 2 macroglobulin
‫الماضيين‬ ‫االتنين‬
HMW
‫في‬ ‫يكتروا‬ ‫لذلك‬
Nephrotic Syndrome
c_ Haptoglobin
‫حاالت‬ ‫في‬
intravascular hemolysis
‫ال‬ ‫في‬ ‫بيمسك‬
Hb
‫الدم‬ ‫في‬ ‫ويقل‬
‫المجموعة‬ ‫هذه‬ ‫معظم‬
HMW
‫حاالت‬ ‫في‬ ‫يزدادوا‬ ‫لذلك‬
Nephrotic Syndrome
‫المجموعتان‬ ‫هاتان‬ ‫معظم‬
Alfa1 and Alfa2 ARE POSITIVE ACUTE PHASE REACTANTS
45

46. B globulin
B globulin ➡
a_ transferrin
‫ال‬ ‫حاالت‬ ‫في‬ ‫بيزداد‬ ‫وده‬
iron deficiency anaemia
b_ C3_C4
c_ B2 microglobulin
d_ hempexin
e_ CRP ➡
‫في‬ ‫جدا‬ ‫جدا‬ ‫يعلي‬
acute infection
f_ protein part of B lipoprotein ( LDL)
g_ hormones as thyroid, estrogen,...
‫ال‬ ‫حاالت‬ ‫في‬ ‫لذلك‬
hyperthyroidism, pregnancy and oral contraceptive pills, B band increased
‫المجموعة‬ ‫هذه‬ ‫معظم‬
HMW
‫حاالت‬ ‫في‬ ‫يزدادوا‬ ‫لذلك‬
Nephrotic Syndrome
Alfa2 and Beta ARE HMW increased in Nephrotic Syndrome
46

47. i.e.
Alfa1 and Alfa2 bands ARE
POSITIVE ACUTE PHASE REACTANTS
Alfa2 and Beta bands ARE
HMW increased in Nephrotic Syndrome
47

48. 48

49. Polyclonal
hypergammaglobulinaemia
¨ Chronic liver disease
¨ Chronic infections
¨ Inflammatory disease of bowel
¨ Autoimmune disorder
¨ Granulomas
•
‫ونلخصها‬
‫بال‬
• Chronic infection and inflammation
49

50. 50

51. Causes of Monoclonal
hypergammaglobulinaemia
‫الجل‬ ‫علي‬ ‫بتعمل‬ ‫دي‬
sharp localized band as :
multiple myeloma
MGUS
waldenstrom macrogloulinaemia
Amylodosis
B cell malignancy as CLL,lymphoma,...
‫بال‬ ‫ونلخصها‬
B cell cancer
51

52. Monoclonal Gammopathies
52

53. Hypogammaglobulinaemia
53

54. Interpretation of electrophoresis :
Interpretation of electrophoresis :
1_ Decreased albumin and globulin band and elevated alfa2 band ( Alfa 2
macroglobulin _ ceruoplasmin ) ➡ indicate Nephrotic Syndrome.
‫ال‬ ‫في‬ ‫ألن‬nephrotic syndrome ‫ال‬ glomeruli ‫لكنن‬ ‫البول‬ ‫في‬ ‫تنزل‬ ‫البروتينات‬ ‫تقوم‬ ‫بتبوظ‬
‫ال‬ ‫نل‬ ‫م‬ ‫الكبينرم‬ ‫البروتيننات‬Alfa 2 macroglobulin ‫هيصننعها‬ ‫كمنان‬ ‫والكبند‬ ‫هتننزل‬ ‫من‬
‫الدم‬ ‫في‬ ‫قلت‬ ‫اللي‬ ‫البروتينات‬ ‫تعويضا‬
.
2_ Increased Alfa 1 and Alfa 2 bands may indicate Acute Phase Reactant.
3_ Increased Alfa 1 band only may indicate chronic hepatitis or Acute Phase Reactant
with hemolytic episode .
4_ Increased alfa 2 band only may indicate immune disease , while decreased alfa 2
band only may indicate hemolytic episode
3_ Increased B band ➡ May indicate estrogen therapy or Iron Deficiency Anaemia
due to increased transferrin .
4_ Protein Loosing Entropathy➡general lowering of all plasma proteins.
5_ Decreased albumin, Alfa 1, Alfa 2, polyclonal gammopathy and fusion of B and
gamma band ➡ indicate Liver Cirrhosis.
54

55. Neohrotic Syndrome
55

56. Acute Infalammation
56

57. Liver Cirrohsis
57

58. IDA
58

59. 59

60. 60

61. ‫ال‬ ‫زي‬ ‫شكل‬ ‫تعمل‬ ‫ممكن‬ ‫حاجات‬ ‫فيه‬
M band
‫زي‬
Bands mistaken for M band :
1_ High fibrinogen in plasma specimen
2_ Hyper Alfa 2 globulinaemia associated with nephrotic syndrome
3_ hyperbetaglobulinaemia as in iron deficiency anaemia
4_ peak at application origin
5_ Hyperlipidaemia
6_ hypergammaglobulinaemia due to excessive immune complex as
high rheumatoid factor activity.
‫أي‬ ‫الخطأ‬ ‫هذا‬ ‫نتفادي‬ ‫لكي‬
Monoclonal band in gamma, beta or even alfa region
‫بنعملها‬
Immunofixation
‫دي‬ ‫بيعرفنا‬ ‫ده‬
M band or not
61

62. 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.
62

63. ‫اليوتيوب‬ ‫علي‬ ‫البحث‬ ‫أو‬ ،‫اليوتيوب‬ ‫علي‬ ‫قناتي‬ ‫دي‬
‫د‬ ‫باسم‬
.
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،
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‫حتى‬ ‫بالقناة‬ ‫ا‬
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DMBMS2018
،
‫حتى‬ ‫تتابعوني‬ ‫ياريت‬
‫هللا‬ ‫شاء‬ ‫إن‬ ‫نستمر‬
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‫هللا‬ ‫شاء‬ ‫إن‬ ‫نستمر‬ ‫حتى‬ ‫تتابعوني‬ ‫ِريت‬‫ا‬‫ي‬
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‫وتعملولي‬ ‫تتابعوني‬ ‫ياريت‬
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