Western blot

Isolation from tissues

ALWAYS ON ICE:

   Use at least 5 mg of tissue;

1. Mince the tissue in a 2 mL tube;

2. Add 300 uL (every 5 mg) of Laemmli lysis buffer + Protease, Phophastase Inhibitors;

3. Add one steel bead/sample;

4. Lysate the tissue using tissue homogenizer (cold room). (3 rounds/5 min each; 1 minute rest). At the end of the procedure, tissue will appear as completely digested. If not, repeat the procedure;

5. Put samples on a rotating platform (cold room) for 2 hours;

6. Centrifuge samples at 14000 rpm (4°C) for 10 minutes;

7. Transfer supernatant in a new tube;

8. Sonicate 15 seconds at 20% amplitude;

9. Boil the samples (95°C);

10. Detect protein concentration;

11. Store at -80°C.

Laemmli Buffer:

WESTERN BLOT

Run:

   Use 30-50 ug of proteins;

1. Dilute proteins with Laemmli Loading Buffer 2x;

2. Boil the samples (95°C), then spin;

3. Load the samples on 4-12% NuPage Gel;

Transfer:

   Use TransBlot mini nitrocellulose system;

1. Take Transfer Chamber, prepare the sandwich (from bottom to top):

   
   

   3 mm paper

   Nitrocellulose

   Gel

   3 mm paper

2. Transfer (7 minutes, use Mixed MW option);

Primary antibody incubation:

1. Pour Ponceau-S solution on nitrocellulose to assess the transfer;

2. Wash 2 minutes with TBS-tween (0.05%v) to get rid of the colour;

3. Block 1 hour (RT) using 5% milk in TBS-t on shaking platform;

4. Incubate primary ab O/N at 4°C on shaking platform.

Protein Detection:

1. Wash membrane 3x with TBS-t (RT), 10 minutes on shaking platform;

2. Incubate secondary antibody 1 hour (RT) on shaking platform;

3. Wash membrane 3x with TBS-t (RT), 10 minutes on shaking platform;

4. Incubate ECL solution for 5 minutes;

5. Detect protein on ChemiDoc.

STRIPPING:

1. Wash the membrane 1x with TBS-t, 10 minutes;

2. Incubate Stripping solution (Invitrogen) for 5-10 minutes on shaking platform;

3. Wash the membrane 1x with TBS-t, 10 minutes;

4. Blocking Step;

5. Incubation with primary ab.

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