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RECIPES > MICROSOMES

> Microsomal Membranes From Dog Pancreas

 

MICROSOMAL MEMBRANES FROM DOG PANCREAS

REAGENTS AND BUFFERS:

All reagents and buffers for microsomes and SRP preps should be made in sterile/baked glassware with water straight from the purifier, and stored in the cold room.
10 x dog


triethanolamine

0.5 M

75 g

potassium acetate

0.5 M

49 g

Mg acetate

0.06 M

12.75 g

EDTA

0.01 M

2.90 g

glacial acetic acid

 until pH 7.6

17.50 mL (approx)

dH2O

 

to 1 litre

 
Dog A (500 mL)
 


10 x dog

1 x

50 mL

2 M sucrose

0.25 M

62.50 mL

dH2O

 

387.50 mL

Dog B Cushions (100 mL)


10 x dog

1 x

10 mL

2 M sucrose

1.1 M

55 mL

dH2O

 

35 mL

Dog C (final resuspension buffer)


2 M TEA pH 7.5

50 mM

2.50 mL

2 M sucrose

250 mM

12.50 mL

dH2O

 

85 mL

Stocks

  1. 2 M sucrose - use 'RNA use only' container!
    171.15 g/250 mL
  2. 2 M triethanolamine (TEA) (pH 7.5)
    Adjust pH with glacial acetic acid, use nuclease free pH meter
  3. 0.1% SDS - Dilute from the 20% stock.  Use this to read the OD of your microsomes.
  4. PMSF (phenylmethyl sulfonyl fluoride)
    Make fresh,
  5. DTT (dithiothreitol) 
    1M DTT is 154 mg in 1 mL dH2O. Store at -20oC. DTT preserves the natural inhibitor of ribonuclease that is present in dog pancreas.  It is because of this inhibitor that we use pancreas from dog (rather than some other animal) to make microsomes.

Use both PMSF and DTT at 1 mM final in all buffers that come into contact with the homogenate.