![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.
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Uploaded byMohammed Bahgat Mohammed Sofyan
Protein Electrophoresis
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.
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Protein Electrophoresis
- 1. PROTEIN ELECTROPHORESIS BY dr: MohammedBahgat Mohammed Sofyan Assistant lecturer of medical biochemistry, Faculty of medicine, Al-Azhar university (Assiut branch) 1
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- 4. صورة كل علىاضغط الشاش عرض وضع في ة قنواتنا على وادعمنا اإلنترنت على
- 5. على ملفي فيهنا أرفعها التي شرائحي ومذاكرة بالتعلم وأسعد وأرحب أسمح Slide share website وال 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 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 themigration 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
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- 9. importance By electrophoresis, amixture of amino acids, proteins, lipoproteins, isoenzyms, CSF proteins, DNA fragments, Hb variant, etc…. can be separated by using electric current. 9
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- 12. PRINCIPLE Proteins are amphotericcompounds 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: containsthe 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
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- 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
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- 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
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- 24. Principle of theProcedure 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 inelectrophoresis 25
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- 29. SUMMARY ال جهاز طريقعن 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
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- 31. UYJ,[[‘0cJJ JUJollectio nيءؤريبرب ريبر Speicment ➡ freshSerum No special prepration is required 31
- 32. UUUUUUU X XZXSASZS Avoid plasmaand 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
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- 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 inelectrophoresis 35
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- 41. hypoalbuminaemia albumin ➡ representabout 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
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- 43. Alfa 1 globulin Alfa1 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
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- 45. Alfa 2 globulin Alfa2 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 Alfa2bands ARE POSITIVE ACUTE PHASE REACTANTS Alfa2 and Beta bands ARE HMW increased in Nephrotic Syndrome 47
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- 49. Polyclonal hypergammaglobulinaemia ¨ Chronic liverdisease ¨ Chronic infections ¨ Inflammatory disease of bowel ¨ Autoimmune disorder ¨ Granulomas • ونلخصها بال • Chronic infection and inflammation 49
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- 51. Causes of Monoclonal hypergammaglobulinaemia الجلعلي بتعمل دي sharp localized band as : multiple myeloma MGUS waldenstrom macrogloulinaemia Amylodosis B cell malignancy as CLL,lymphoma,... بال ونلخصها B cell cancer 51
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- 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
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- 61. ال زي شكلتعمل 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 البحثأو ،اليوتيوب علي قناتي دي د باسم . سفيان بهجت محمد ، وتشتركو تتابعوني ياريت حتى بالقناة ا هللا شاء إن نستمر https://www.youtube.com/channel/UCaYs1d8s0ntZvteHS3mMmGA ب الفيس في البحث أو ،انضمامكم يشرفنا الفيسبوك علي بتاعتي الصفحة دي DMBMS2018 ، حتى تتابعوني ياريت هللا شاء إن نستمر https://www.facebook.com/DMBMS2018/ باسم التلجرام علي قناتي ودي ِ للناس أنفعهم الناس خير 📚 هللا شاء إن نستمر حتى تتابعوني ِريتاي https://t.me/DMBMS2020 على بروفايلي لينكيدإن وتعملولي تتابعوني ياريت endorsement مهاراتي على https://www.linkedin.com/in/mohammed-bahgat-sofyan-8ba012142/ موقع على بروفايلي وده SlideShare تتابعونا ياريت https://www.slideshare.net/MohammedBahgatMohamm1 وده بروفايلي الشخصي الفيسبوك علي https://www.facebook.com/mohammed.bahgat.165 الفيسبوك على بنا الخاص اللوجوا وده # دمحمد _ بهجت _ سفيان _ medical_biochimestry 63
- 64. My logo onFacebook # دمحمد _ بهجت _ سفيان _ medical_biochimestry 64
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