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^J #e l l s a n d r e l e a s e o f i n t r a c e l l u l a r c o m p o n e n t s , c e n t r i f u g a t i o n 2 0 - m i n a t t h e s p e e d o f 2 0 0 0 - 3 0 0 0 r . p . m . r e m o v e s u p e r n a t a n t , I f p r e c i p i t a t i o n a p p e a r e d , C e n t r i f u g a l a g a i n .
T i s s u e s a m p l e s - A f t e r c u t t i n g s a m p l e s , c h e c k t h e w e i g h t , a d d P B S P H 7 . 2 - 7 . 4 , R a p i d l y f r o z e n w i t h l i q u i d n i t r o g e n , m a i n t a i n s a m p l e s a t 2 - 8 ! a f t e r m e l t i n g , a d d P B S P H 7 . 4 , H o m o g e n i z e d b y h a n d o r G r i n d e r s , c e n t r i f u g a t i o n 2 0 - m i n a t t h e s p e e d o f 2 0 0 0 - 3 0 0 0 r . p . m . r e m o v e s u p e r n a t a n t .
e x t r a c t a s s o o n a s p o s s i b l e a f t e r S p e c i m e n c o l l e c t i o n , a n d a c c o r d i n g t o t h e r e l e v a n t l i t e r a t u r e , a n d s h o u l d b e e x p e r i m e n t a s s o o n a s p o s s i b l e a f t e r t h e e x t r a c t i o n . I f i t c a n t , s p e c i m e n c a n b e k e p t i n - 2 0 ! t o p r e s e r v e , A v o i d r e p e a t e d f r e e z e - t h a w c y c l e s .
C a n t d e t e c t t h e s a m p l e w h i c h c o n t a i n N a N 3 , b e c a u s e N a N 3 i n h i b i t s H R P a c t i v e .
A s s a y p r o c e d u r e
1 . N u m b e r : t o s a m p l e c o r r e s p o n d m i c r o t i t r a t i o n w e l l a n d N u m b e r S e q u e n c e , e a c h p l a t e s h o u l d b e s e t f e m i n i n e c o m p a r i s o n 2 w e l l s , m a s c u l i n e c o m p a r i s o n 2 w e l l s , b l a n k c o m p a r i s o n 1 w e l l ( d o n t a d d s a m p l e a n d H R P - C o n j u g a t e r e a g e n t t o b l a n k c o m p a r i s o n w e l l , o t h e r e a c h s t e p t h e o p e r a t i o n a r e s a m e ) .
2 . a d d s a m p l e s e p a r a t e l y a d d P o s i t i v e c o n t r o l a n d N e g a t i v e c o n t r o l 5 0 l t o t h e P o s i t i v e a n d N e g a t i v e w e l l . a d d S a m p l e d i l u t i o n 4 0 l t o t e s t i n g s a m p l e w e l l , t h e n a d d t e s t i n g s a m p l e 1 0 l . a d d s a m p l e t o t h e b o t t o m o f E L I S A p l a t e s c o a t e d w e l l , d o n t t o u c h t h e w e l l w a l l a s f a r a s p o s s i b l e , a n d G e n t l y m i x .
3 . I n c u b a t e : A f t e r c l o s i n g p l a t e w i t h C l o s u r e p l a t e m e m b r a n e , i n c u b a t e f o r 3 0 m i n a t 3 7 !.
4 . C o n f i g u r a t e l i q u i d : 3 0 - f o l d ( o r 2 0 - f o l d ) w a s h s o l u t i o n d i l u t e d 3 0 - f o l d ( o r 2 0 - f o l d ) w i t h d i s t i l l e d w a t e r u n t i l 6 0 0 m l , a n d r e s e r v e .
5 . w a s h i n g U n c o v e r C l o s u r e p l a t e m e m b r a n e , d i s c a r d L i q u i d , d r y b y s w i n g , a d d w a s h i n g b u f f e r t o e v e r y w e l l , s t i l l f o r 3 0 s t h e n d r a i n , r e p e a t 5 t i m e s , d r y b y p a t .
6 . a d d e n z y m e A d d H R P - C o n j u g a t e r e a g e n t 5 0 l t o e a c h w e l l , e x c e p t t h e b l a n k w e l l .
7 . i n c u b a t e O p e r a t i o n w i t h 3 .
8 . w a s h i n g O p e r a t i o n w i t h 5 .
9 . c o l o r A d d C h r o m o g e n S o l u t i o n A 5 0 u l a n d C h r o m o g e n S o l u t i o n B t o e a c h w e l l , e v a d e t h e l i g h t p r e s e r v a t i o n f o r 1 5 m i n a t 3 7 !
1 0 . S t o p t h e r e a c t i o n A d d S t o p S o l u t i o n 5 0 l t o e a c h w e l l , S t o p t h e r e a c t i o n ( t h e b l u e c o l o r c h a n g e t o y e l l o w c o l o r ) .
1 1 . a s s a y t a k e b l a n k w e l l a s z e r o , R e a d a b s o r b a n c e a t 4 5 0 n m a f t e r A d d i n g S t o p S o l u t i o n a n d w i t h i n 1 5 m i n .
D e t e r m i n e t h e r e s u l t
T e s t v a l i d i t y : t h e a v e r a g e o f P o s i t i v e c o n t r o l w e l l e"1 . 0 0 ; t h e a v e r a g e o f N e g a t i v e c o n t r o l w e l l d"0 . 1 0 .
C a l c u l a t e C r i t i c a l ( C U T O F F ) : C r i t i c a l = t h e a v e r a g e o f N e g a t i v e c o n t r o l w e l l + 0 . 1 5 .
N e g a t i v e c o n t r o l : s a m p l e O D < C a l c u l a t e C r i t i c a l ( C U T O F F ) i s B V D A b N e g a t i v e c o n t r o l .
P o s i t i v e c o n t r o l : a m p l e O D e" C a l c u l a t e C r i t i c a l ( C U T O F F ) i s B V D A b P o s i t i v e c o n t r o l .
I m p o r t a n t n o t e s
1 . P l e a s e a c c o r d i n g t o u s e i n s t r u c t i o n s t r i c t l y , D o n o t m i x r e a g e n t s w i t h t h o s e f r o m o t h e r l o t s .
2 . T h e k i t t a k e s o u t f r o m t h e r e f r i g e r a t i o n e n v i r o n m e n t s h o u l d b e b a l a n c e d 1 5 - 3 0 m i n u t e s i n t h e r o o m t e m p e r a t u r e t h e n u s e , E L I S A p l a t e s c o a t e d i f h a s n o t u s e u p a f t e r o p e n e d , t h e p l a t e s h o u l d b e s t o r e d i n S e a l e d b a g .
3 . w a s h i n g b u f f e r w i l l C r y s t a l l i z a t i o n s e p a r a t i o n , i t c a n b e h e a t e d t h e w a t e r h e l p s d i s s o l v e w h e n d i l u t e . W a s h i n g d o e s n o t a f f e c t t h e r e s u l t .
4 . C l o s u r e p l a t e m e m b r a n e o n l y l i m i t s t h e d i s p o s a b l e u s e , i n o r d e r t o a v o i d t h e o v e r l a p p i n g p o l l u t i o n
5 . T h e s u b s t r a t e p l e a s e e v a d e t h e l i g h t p r e s e r v a t i o n .
6 . T h e t e s t r e s u l t d e t e r m i n a t i o n m u s t t a k e t h e m i c r o t i t e r p l a t e r e a d e r a s a s t a n d a r d , w h e n u s e d u a l - w a v e l e n g t h t o a s s a y , R e f e r e n c e w a v e l e n g t h i s 6 3 0 n m .
7 . A l l s a m p l e s , w a s h i n g b u f f e r a n d e a c h k i n d o f r e j e c t s h o u l d a c c o r d i n g t o i n f e c t i v e m a t e r i a l p r o c e s s . S t o p p S o l u t i o n i s 2 M s u l p h u r i c a c i d . Y o u m u s t p a y a t t e n t i o n t o s a f e w h e n u s e .
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