UCSF

Protein Extraction

Extract protein from tissue or cell lines

9/30/2009 Maggie

Prepare ice! During the extraction procedure, the protein should always be kept on ice once it’s been put into the extraction buffer and later on in order to avoid degradation and keep activity.

For fresh or frozen tissue

  • 10% bleach, 100% ethanol, dH2O
  • Homogenizer

1. Prepare 10% bleach, 100% ethanol, dH2O for cleaning the Homogenizer. No more than 5ml/tube.

For 6 samples, prepare 2+6+2=10 tubes of each cleaning solution. 10× 5ml=30ml.

For 8 samples, prepare 2+8+2=12 tubes of each cleaning solution. 12× 5ml=60ml.

First wash, each Twice (i.e. 10% bleach→10% bleach→100% ethanol→100% ethanol→ H2O→H2O).

Between each sample, wash once (i.e. 10% bleach→100% ethanol→ H2O).

Last wash, each Twice (i.e. 10% bleach→10% bleach→100% ethanol→100% ethanol→ H2O→H2O).

For cell line (Step1, 2 then go to step 6)

1 Carefully removes culture medium. Wash the cell with PBS twice gently.

Add 1 ml PBS into the well, scrap the cell from the plate.

Spin, RT or 4°C, 3000rpm, 1-3 min.

2. Prepare extraction reagent. (The same reagent for cell line protein extraction)

Each tissue

× 7

× 9

6-well plate

Each well

 

M-PER Reagent

300 μl

2100 μl

2700 μl

50 μl

Protease and Phosphatase Inhibitor

3UL

21 μl

27 μl

0.5 μl

         

M-PER® Mammalian Protein Extraction Reagent: Pierce, Cat.78503, purple vial, stored at 4°C.

Halt™ Protease and Phosphatase Inhibitor Cocktail: Thermo, Cat.78440, white bottle, stored at 4°C.

Aliquot the reagent mixture into plastic tubes.

3 Prepare dry ice.

Take out the Eppendorf containing the tissues from -80°C, put a pieces of tissue with a pipette tip into the plastic tube containing the extraction reagent. Put the Eppendorf on the dry ice after use.

4 Homogenize tissues

Set the homogenizer to 30.

Wash as step1 mentioned.

Homogenize each sample for about 20-30 seconds. (Remember to wash the homogenizer after each sample homogenizing.)

5 Transfer to Eppendorf tubes and incubate on ice for 10 minutes.

(Meanwhile, put the Eppendorf containing the tissues back to -80°C.)

6 Spin at 4°C, 13000rpm for 10 min.

7 Prepare the sample buffer in the hood.

Usually 250 ul buffer / tissue sample.

Usually 50 ul buffer / tissue sample.

(For 100 μl: 95ul of Laemmli Sample buffer (Bio-Rad, blue, store at RT) + 5 ul of β-ME).

For tissue

 

Each sample

       
 

100 ul

250 ul

× 6

× 8

× 10

Laemmli Sample buffer

95

237.5

1425

1900

2375

β-ME

5

12.5

75

100

125

           

For cell line

 

Each sample

     
 

100 ul

50 ul

× 6

× 8

× 10

Laemmli Sample buffer

95

47.5

285

380

475

β-ME

5

2.5

15

20

25

Aliquot into labeled Eppendorf.

9. Add same volume of supernatant into the Eppendorf with the sample buffer.

10. Incubate at >70°C (70-100°C) at heat block for 5 min. (Remember to cover the tube cap)

11. Store the samples at -80°C.