Molecular biology of the cell nucleus 1

Analysis of chromatin strucutre in human cell nuclei

How to make final target concentrations from a stock solution

e.g 10ml of 10mM from a 1M

1. Work out dilution factor: 1/0.01= 100

2. Divide target volume 10ml by dilution factor

3. take this volume from stock solution

4. fill rest with water until it is 10ml (add difference in volume)

If the colution contrains more than one component

• you need to add the required volumes for each component successively

• then adjust the remaining amount of water accordingly

Prepareing the cell nucleui

1. ake hypotonic cell resuspension buffer

2. make cell lysis buffer

3. Remove supernatent from centrifuged HeLa cells

4. add cold hypotonic cell resuspension buffer

5. resuspend up  and down with pipette but gently→ do not break or shear 

6. Add cell supension to two tubes A and B, put on ice

7. Add cold cell lysis buffer to tube A + resuspend

   1. non-ionic detergenet (NP40) will lyse cellular membranes and leave nuclei enclosed in their lamina

   2. put back on ice

   3. Spin A and B tube on low sped

   4. pellets the nuclei

   5. remove supernatants and ice

EXPERIMENT 1: Analysis of nuclear proteins by denaturing polyacrylamide gel electrophoresis

1. Take A add protein sample buffer

   1. it contains SDS, beta-mercaptoethanol and bromophenol blue as a dye

   2. resuspend pelleted nuclei up and down

   3. heat for 10 mins with cap off

      1. denature proteins and nucleic acids, polypeptides can be separated on polyacrylamide gel

      2. ionic detergent SDS binds to denatured protein and adds a large negative charge to surface→ allow for gel electro separation (so now based on weight)

   4. Close lid and leave at sample room temp

   5. vortex→ shear genomic DNA and reduces

   6. load onto gel

EXPERIMENT 2: Analysis of chromatin strucuture: outline

• Analyse the strucutre of chromatin as found in human cell nuclei

• treat cells with endonuclease to cut DNA accessible within the chromatin to the nuclease

  • mainly just DNA that links adjacent nucleosomes

• prepare culease-resistant DNA fragments

• separate them according by length by neutral agarose gel electrophoresis

Experiment 2: Preparation

1. Add Tris-Cl pH8 buffer and CaCl2 to the cells

   • Resuspend

   • → washes the nuclei

   • AND→ Ca2+ needed for the nuclease (added later)

2. Spin on low speed

3. remove supernantant and resuspend nuceli by tapping the sides

4. Warm to room temp

5. Thaw frozen Micrococcal Nuclease and add

6. incubate for set time (1,3,6,or 9 min)→ at room temp

7. STOP reaction with DNA sample buffer and phenol:chloroform

8. vortex immediately

Experiment 2: why does the DNA buffer and Phenol: chloropform stop the nuclease activity

1. DNA buffer:

• nuclease needs Ca2+ for activity

• EDA agent in the DNA buffer (sol H) chelating agent

also

• contains strong ionic detergenet SDS → denature proteins

2. Phenol: chloroform

   • irreverably denatures proteins and precipitates them out

   • including nuclease

MOST IMPORTANTLY: does not denature nucleic acids

How does the phenol denature proteins?

• Alters solubility

1. forces protein to unfold

2. exposing its hydrophobic redsidues

3. so now less soluble in water

Experiment 2: Contiued preparation (after stopping reaction)

1. Spin sample of high: this forms

   • UPPER PHASE→ aqueous with nucleic acids

   • Middle→ proteins (white interphase)

   • LOWER→ Phenolic phase (hydrophobic)

2. Only want the UPPER PHASE with nucleic acids

3. Add phenol:cholroform again, vortex and spin again and take upper phase

4. Add sodium acetate and ethanol

5. Freeze sample for 10 mins

6. Centriuge on high for 10 mins

What will this acetate, ethanol freezing treatment do?

• precipicate the nucleic acids

• so can be isolated from the reaction by centrifugation

Experiment 2: continued (after centrifugation)

1. discarad supernantant

2. ensure all ethanol off the SNA sample→ because will not dissolve other wise

3. Add DNA smaple buffer and dissolve

4. Load onto 2% agarose gel

Place make a diagram, flow chart of these experiments

(instead)