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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 .
A s s a y p r o c e d u r e
1 . D i l u t e a n d a d d s a m p l e t o S t a n d a r d : s e t 1 0 S t a n d a r d w e l l s o n t h e E L I S A p l a t e s c o a t e d , a d d S t a n d a r d 1 0 0 l t o t h e f i r s t a n d t h e s e c o n d w e l l , t h e n a d d S t a n d a r d d i l u t i o n 5 0 l t o t h e f i r s t a n d t h e s e c o n d w e l l , m i x ; t a k e o u t 1 0 0 l f o r m t h e f i r s t a n d t h e s e c o n d w e l l t h e n a d d i t t o t h e t h i r d a n d t h e f o r t h w e l l s e p a r a t e l y . t h e n a d d S t a n d a r d d i l u t i o n 5 0 l t o t h e t h i r d a n d t h e f o r t h w e l l , m i x ; t h e n t a k e o u t 5 0 l f r o m t h e t h i r d a n d t h e f o r t h w e l l d i s c a r d , a d d 5 0 l t o t h e f i f t h a n d t h e s i x t h w e l l , t h e n a d d S t a n d a r d d i l u t i o n 5 0 l t o t h e f i f t h a n d t h e s i x t h w e l l , m i x ; t a k e o u t 5 0 l f r o m t h e f i f t h a n d t h e s i x t h w e l l a n d a d d t o t h e s e v e n t h a n d t h e e i g h t h w e l l , t h e n a d d S t a n d a r d d i l u t i o n 5 0 l t o t h e s e v e n t h a n d t h e e i g h t h w e l l , m i x ; t a k e o u t 5 0 l f r o m t h e s e v e n t h a n d t h e e i g h t h w e l l a n d a d d t o t h e n i n t h a n d t h e t e n t h w e l l , a d d S t a n d a r d d i l u t i o n 5 0 l t o t h e n i n t h a n d t h e t e n t h w e l l , m i x , t a k e o u t 5 0 l f r o m t h e n i n t h a n d t h e t e n t h w e l l d i s c a r d ( a d d S a m p l e 5 0 l t o e a c h w e l l a f t e r D i l u t i n g , ( d e n s i t y : 4 8 0 p g / m l 3 2 0 p g / m l 1 6 0 p g / m l 8 0 p g / m l 4 0 p g / m l )
2 . a d d s a m p l e S e t b l a n k w e l l s s e p a r a t e l y ( b l a n k c o m p a r i s o n w e l l s 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 , o t h e r e a c h s t e p o p e r a t i o n i s s a m e ) . t e s t i n g s a m p l e w e l l . a d d S a m p l e d i l u t i o n 5 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 ( s a m p l e f i n a l d i l u t i o n i s 5 - f o l d ) , a d d s a m p l e t o w e l l s , 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 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 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 .
I m p o r t a n t n o t e s
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 , 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 .
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 .
a d d S a m p l e w i t h s a m p l e r E a c h s t e p , A n d p r o o f r e a d i t s a c c u r a c y f r e q u e n t l y , a v o i d s t h e e x p e r i m e n t a l e r r o r . a d d s a m p l e w i t h i n 5 m i n , i f t h e n u m b e r o f s a m p l e i s m u c h , r e c o m m e n d t o u s e V o l l e y .
i f t h e t e s t i n g m a t e r i a l c o n t e n t i s e x c e s s i v e l y h i g h e r ( T h e s a m p l e O D i s b i g g e r t h a n t h e f i r s t s t a n d a r d w e l l ) , p l e a s e d i l u t e S a m p l e ( n - f o l d ) , P l e a s e d i l u e n t e a n d m u l t i p l i e d b y t h e d i l u t i o n f a c t o r . n 5 .
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 , t o a v o i d c r o s s - c o n t a m i n a t i o n .
T h e s u b s t r a t 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 .
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 , 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 .
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 .
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 .
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2 v a l i d i t y s i x m o n t h s .
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T a k e t h e s t a n d a r d d e n s i t y a s t h e h o r i z o n t a l , t h e O D v a l u e f o r t h e v e r t i c a l , d r a w t h e s t a n d a r d c u r v e o n g r a p h p a p e r , F i n d o u t t h e c o r r e s p o n d i n g d e n s i t y a c c o r d i n g t o t h e s a m p l e O D v a l u e b y t h e S a m p l e c u r v e , m u l t i p l i e d b y t h e d i l u t i o n m u l t i p l e , o r c a l c u l a t e t h e s t r a i g h t l i n e r e g r e s s i o n e q u a t i o n o f t h e s t a n d a r d c u r v e w i t h t h e s t a n d a r d d e n s i t y a n d t h e O D v a l u e , w i t h t h e s a m p l e O D v a l u e i n t h e e q u a t i o n , c a l c u l a t e t h e s a m p l e d e n s i t y , m u l t i p l i e d b y t h e d i l u t i o n f a c t o r , t h e r e s u l t i s t h e s a m p l e a c t u a l d e n s i t y .
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