Protein extraction and Macf1 identification by western blot

Protein extraction and Macf1 identification by western blot

This protocol describes detailed steps for the identification of mouse Macf1 protein by western blot. Since mouse Macf1 is a huge protein with two main isoforms at 830kD and 630kD, this protocol can be used/adjusted for the detection of similar large proteins.

Protein extraction and dosage:

1. Prepare RIPA lysis buffer by adding the following components to purified water in order to obtain the following final concentrations:

   a. 10mM Tris HCl, pH=7.5-8

   b. 150mM NaCl

   c. 0.5mM EDTA

   d. 0.1% SDS

   e. 1% Triton X-100

   f. 1% Sodium Deoxycholate

    

2. Complete the lysis buffer with phosphatase inhibitors (Sigma, P5726-5mL) and protease inhibitors (Sigma, P8340) as instructed by the manufacturer. Always keep the final lysis buffer on ice.

    

3. Depending on the sample type and size, add the proper amount of the lysis buffer to coated cells on a culture dish (note that cells can be scrabbed directly in the lysis buffer), dried cell pellet, grounded/powdered organ, or organ chops on ceramic beads (for example, MP Biomedicals Lysing Matrix D, 6913100. Note that organs can be subjected to bead-mediated break directly in the lysis buffer).
    

4. Keep the lysis tubes on ice, for at least 30 minutes, and vortex frequently.
    

5. Subject the samples to 5 cycles of low sonication (cycles of 30 seconds ON, 30 seconds OFF) using a Bioruptor sonication device (Diagenode, B01020001) or equivalent.
    

6. Centrifuge the samples for 15 minutes at 12500RPM and 4°C.
    

7. Collect the supernatant into a fresh tube and discard the pellet. Samples can be used directly for dosage and/or kept at -20°C until used.
    

8. Use Pierce™ BCA Protein Assay Kits (ThermoFisher Scientific, 23225) or equivalent, to determine the protein concentration for each sample as instructed by the manufacturer.

Protein detection by western blot:

1. Manually cast the western blot Acrylamide gel in order for the final concentration of the Acrylamide to be at 4% in both running and stacking sections of the gel. The following recipe is given for the preparation and casting of a single gel (1.5mm of thickness):

1. 10mL of running gel with 4% Acrylamide:
   1. 4.712mL of H2O
   2. 1.33mL of 30% Acrylamide
   3. 3.75mL of 1M Tris, pH 8.8
   4. 100μL of 10% SDS
   5. 100μL of 10% APS
   6. 8μL of TEMED
2. 5mL of stacking gel with 4% Acrylamide:
   1. 3.599mL of H2O
   2. 666μL of 30% Acrylamide
   3. 630μL 1M Tris, pH 6.8
   4. 50μL of 10% SDS
   5. 50μL of 10% APS
   6. 5μL of TEMED

2. Prepare the samples for loading, by mixing 60μg of the extracted protein per condition with the proper amount of the Laemmli buffer and H2O to have the Laemmli buffer diluted to 1X and the same loading volume for every sample.

3. When the gel is polymerized, mount the glasses onto the clamping frame, and fill the electrophoresis tank with the 1X Tris-Glycine-SDS migration buffer.

4. Load the samples to desired wells. We recommend using HiMark™ Pre-Stained Protein Standard (Invitrogen, LC5699) to follow the migration.

5. Run the gels on a low voltage of 130V for around 10 minutes, until samples enter the gel, and increase the voltage to 170V for 90 to 120 minutes, until the 460kD band of the Pre-Stained Protein Standard reaches the middle of the running gel (or lower).

6. When the migration is done, mount the gel-membrane sandwich using a 0.45μm nitrocellulose membrane into the gel holder cassettes.

7. Fill the transfer tank with the proper amount of the transfer buffer. To prepare the transfer buffer add the following components to purified water in order to obtain the following final concentrations:

1. 1X Tris-Glycine
2. 20% of Ethanol
3. 0.1% of final SDS

8. Transfer the protein to the nitrocellulose membrane, overnight (18 hours) at 60V and 4°C.

Note 1: to ensure a stable temperature, it is recommended to run the transfer in a cold room or cover the transfer bucket with a sufficient amount of ice that would be stable overnight).

Note 2: to avoid precipitate formations in the transfer buffer, it is recommended to use a magnetic stirrer set at 300RPM directly in the transfer tank.

9. Following the transfer, wash the membrane once with H2O.

10. Block the membrane 5% milk-1X TBS for 1H at RT.

11. Incubated the membrane in Macf1 primary antibody (Novus Biologicals, NBP2-36528) diluted 1/1000 in 1% milk-1X TBS over night at 4°C.

12. Wash the membrane 3 times using 0.1% Tween 20-1 X TBS.

13. Incubate the membrane in anti-rabbit secondary antibody (Invitrogen, 65–6120) diluted 1/5000 in 5% milk-0.1% Tween 20-1X TBS for 1H at RT.

14. Wash the membrane 3 times using 0.1% Tween 20-1 X TBS.

15. Use SuperSignal™ West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific, 34096) following the manufacturer's instruction to detect the protein bands.