Sds page electrophoresis

SDS-PAGE is a technique used to separate proteins .
The concept of stacking and resolving gel in this SDS-PAGE are briefly describe in very interesting and easy way to understand the role of stacking and resolving gel.

1. P r e s e n t e d b y - K u m a r i Jyot i M s c i n b i o t e chnology a n d diploma i n f o r e n s ic science V i n o b a b h a v e u n i v e rsity Topics - S D S - P A G E

2. S o d i u m d o d e c y l sulphate - poly a c r y l a m i d e g e l electrophoresi s . ( S D S - P A GE )

3. P r i n c i p l e M a t e r ial a n d method' s Applicatio n s

4. P r i n c i p l e o f SDS-PAGE S D S - E l e c t r o p h o r e s i s i s a t e c h n i q u e u s e d t o s e p a r a t e p r o t e i n m o l e c u l e s according t o molecular wright . P r o t e i n i s n e g a t i v e l y c h a r g e d ( d u e t o S D S d e t e r g ent ) a n d w h e n e l e c t r i c f i e l d i s a p p l i e d a c ross t h e g e l , p r o t e in m i g r a t e s t h r o u g h t h e g e l i s d e p e n d e n t u p o n - Mol ecular w e i g ht o f p r o t e i n A garose- concentrations A p p l i e d c u rrent

5. M a t e r ial s u s e d i n S D S e l e c t r o p h o r e s i s a n d i t s f u n c tio n .

6. P o l y a c r y l a m i d e g e l - Reason behind p o l y acrylamide u s e d i s - I t i s i n e r t E l e c t r i c a lly n e u t ral T r a n s p a r ent C r o s s l i n k e d p o l y a c r ylamide g e l a r e formed b y c o - p o l y m e r ization o f a c r y l a m i d e m o n o m e r a n d a c r o s s l i n k i ng b y N , N ' - M e t h y l e n e - b i s - a c r y l a m i d e . A m m o n i u m p e r s u l p h a te i s used a s initiator . T E M E D i s used a s c a t a l y s t . L o w e r p e r c e n t a g e o f g e l s - f a v o u r l a r g e r p o r e s i z e - i t separate l a r g e r molecules H i g h e r percentage o f g e l - favour s m a l l e r p o r e s i z e - i t s e p a r a te smaller m o l ecules .

7. B u f f e r - SDS - P A G E u t i l i s e discontinuous b u f f e r s y s t e m . B u f f e r s y s t e m s i n c l u de t h e b u f f e r used t o - C a s t t h e g e l Prepare t h e sample R u n n i n g b u f f er p r e p a r ation

8. B u f f e r s u s e d i n S D S - P A G E a r e - T r i s glycine ( pH = 8 . 3 ) T r i s HCl ( pH = 6 . 8 ) - T r i s H C l ( pH = 8 . 8 ) - Tris g l y c i n e ( pH = 8 . 3 ) i s used i n r u n n ing buffer . T r i s HCl ( pH = 6 . 8 ) used i n s t a c k ing g e l Tris HCl ( pH = 8 . 8 ) i s u s e d i n s e p a r a t i ng g e l .

9. S D S - S o d i u m d o d e c y l s u l p hate i s a n anionic d e t e r g e nt . I t b i n d s s t r o n g l y t o p r o t e i n s , c a u s i n g t h e i r denaturation . SDS m a k e t h e p r o t e in negatively c h a r g ed .

10. B e t a m a r c a p t o e t h a n o l - I s used t o c l e a v e s t h e d i s u l f i d e b o n d s . B r e a k s t h e d i s u l p h ide b o n d p r e s e n t i n p r o t ein P r o t e i n

11. Steps i n S D S -PAGE

12. S t e p s - G e l p r e p a r a t i on . Casting t h e g e l . L o a d i n g t h e samples Observ e the protein b a n d s R e s u l t s interpretation . S t a c k i ng gel S e p a r a t i n g g e l

13. P o l y a c r ylamide g e l p r e p a r a t i o n f o r s t a c k i n g g e l A c r y l a m i d e - l e s s c o n c e n t r a t i o n . 5 M Tris ( pH = 6 . 8 ) S D S d H 2 O A P S ( a m m o n i um p e r s u l p hate ) T E M E D

14. P o l y a c r ylamide g e l p r e p a r a t i o n f o r r e s o l v i n g g e l / s e p a r a t i n g g e l A c r y l a m i d e - h i g h c o n c e n t r a t i o n 1 . 5 M Tris ( pH = 8 . 8 ) S D S d H 2 O A P S ( a m m o n i um p e r s u l p hate ) T E M E D

15. F i r s t l y we p o u r t h e resolving g e l i n g e l c a s t e r A f t e r t h e g e l i s s e t w e place t h e c o m b i n c a s t e r A fter t h e g e l i s s e t w e p o u r the s t a c k i ng g e l a b o v e t h e resolving g e l o r separating g e l . A d d r u n n i ng b u f f e r t o c o v e r t h e s u r f a c e o f t h e g e l .

16. Sample preparation

17. B r o m o p h e n o l b l u e d y e r o l e - I t i s u s e d t o t r a c k t h e p r o t e in i n g e l d u r i ng e l e c t r ophoresis

18. I n v i s i ble ( protein i s co l urless ) Treated with d y e Protein i s v i s u alize

19. D y e i s u s e d t o v i s u alize t h e p r o t e i n j u s t like t h e M r India i n v i s i b le concept - W h e n i n v i s i b le w a t c h i s activated i t make t h e w e a r e r i n v i s i b le t o n a k e d e y e b u t w h e n red l i g h t i s f o c u sed o n the w e a r e r , t he p e r s o n b e c o m e visible .

20. S D S ( a n i o n i c d e t e r g e n t ) make t h e p r o tein n e g a tively c h a r g e d P r o t ein

21. B e t a m a r c a p t o e t h a n o l - r o l e

22. W i t h o u t g l y c e r ol t h e sample i s l e s s dense s o i t i s not settle d i n w e l l G l y c e r ol p r o v i de t h e d e n s i ty t o t h e sample

23. R o l e o f s t a c k i ng g e l

24. S t a c k i ng gel p l a y a r o l e t o s t a c k protein a t o n e position .

25. S t a c k i n g g e l i s u s e d t o s t a c k t h e p r o t ein i n o n e p l a c e s o t h a t they a l l e n t e r i n r e s o l v ing g e l at s a m e t i m e , j u s t like the m a r a t h o n e r s s t a c k i n o n e p o sition b e f o r e t h e run .

26. R o l e o f s e p a r a ting gel

27. R e s o l v i ng g e l also c a l l ed separation g e l i s u s e d t o separate protein m o l ecules a c c o rding t o their m a s s .

28. F i g u re s h o w t h a t sample e n t e r i n s e p a r a t i n g g e l a n d C h l o rine r e a c h e s t h e i s t p o s i tion G l y c i ne i n s e c o n d Protein i s t h i r d position

29. S t a i n i ng dye - Add t h e c o o m a s s i e b l u e t o visualize t h e bands w i t h c l e a r background .

30. G e l d o c s y s t e m i s used t o v i s ualize t h e P r o tein bands .

31. R e s u l t - p rotein b a n d s

32. V i d e o l i n k for SDS P A G E https://youtu.be/eaETFKXtNRA

33. A p p l i cations o f S D S - P A G E I t i s u s e d t o m e a s u r e t h e m o l ecular w e i g h t of t h e molecules . I t i s u s e d i n p e p t i de m a p p i n g I t i s u s e d t o e s t i m ate p u r i ty o f p r o tein s . I t i s u s e d f o r s a m p l e p r e p a ration i n p r i o r t o s p e c t r o metry

34. T h a n k y o u