Western blot

Protein extraction

1-5 x10^6 BMDC are used per condition.

Place the cell in security tubes (the ones with the rubber in the cap) in warm media.

Add the treatments

Activate the cells at 37C (activation time would depend of the protein that will be detected).

For MAPK (JNK, ERK and p38) 15 minutes of activation it is good time to see phosphorylation.

I recommend to activated the cells in the water bath or place them in the C02 incubator, after activation.

After this time, cells are collected by centrifugation 5 min 14,000. If you can centrifuge at 4C it would be better (cold will stop the reaction).

Extract the supernatant with a vacuum or a pipette, try not to touch the cellular pellet.

Lysate the cellular pellet using 200 ul of Laemmbli 1X boiled, complemented with 50ul of B-mercaptoethanol per ml of Laemmbli 1X. Laemmbbli could be diluted in distilled water.

Boil the samples for 10 minutes and vortex the samples every 3 minutes.

Keep the samples at -80C

Running gel

Prepared the gel or use the gels from Genscript, Express Plus PAGE gel, this company specified that these gels have to run in MOPS buffer and not in TRIS-Glycine buffer. We have MOPS buffer in powder. The percentage of the gel will depend of the weight of the proteins to analyze. In our case our gels are 12% (Standard gel, for media size proteins).

Run the gel for 1-1.5 hrs, in cold room or inside in ice bucket. For the molecular marker use only 5ul to load in the gel, load 40ul for samples. Check the running of the bands every 20 minutes, if the gel it is running too slow, increase the current. Also check for buffer in the inner chamber for leaks. Voltage is around 80 to 100V.

Transfer the gel.

For transfer the gel follow the instructions provided by the XCell blot module by Invitrogen.

Prepare the transfer buffer 1X, and add methanol. Soak the sponges in the transfer buffer. Assemble the gel/membrane sandwich. Place the sandwich unit in the chamber (do not forget to clean the chamber with water to remove the running buffer), add transfer buffer in the inner chamber, check for any leaks. Add water in the outside. Place the chamber in an ice bucket or do the transfer in the cold room. Transfer using 25-35 volts for 1-1.5 hrs. During this time, check the buffer level in the inner chamber for leaks. In case of leak, turn off the power supply and add more transfer buffer in the inner chamber.

Blocking.

Block each membrane with 25 ml of 5% non-fat dry milk in TBS-T for 1 hr at RT in a shaker, after this time, wash the membrane 2 times for 5 minutes in 25 ml of TBS-T using the shaker.

After this, the membrane it is ready to be incubated with the antibody.

Incubation.

Depending of the antibody and the protein it will be the dilution, usually 1:1000, or 1:2000 are good to try. The antibody dilution it is made in TBS-T. Use bags to save antibody; you can place two membranes in the same bag. 5 ml it is enough to cover the membranes. Place the membrane in a shaker at 4C, overnight.

Developing.

Remove the antibody from the bag, sometimes it is worth to collect the antibody to re-use it. It is the case collect the antibody in a clean tube and keep it at 4C. Wash the membrane two times with TBS-T for 5minutes., use the shaker at RT. After this time, Incubate the membrane with the secondary antibody, the dilution of this antibody have to be standardized but usually 1:10,000 it is good to start. Use 10 ml to incubate the membrane, in the shake at RT for 1 hr. After this time, wash the membrane, 3 times with 25 ml for 10 minutes (this time it have to be standardized). After this develop the membrane following the instruction for the developing kit.

Exposure

Exposure the membrane in the cassette for the time necessary to detect the signal.

TBS 10X 1 L

Tris 60.6 gr

NaCl 80.6 gr

1 L distilled water

Adjust the pH 7.4

TBS-T

TBS 1X plus 0.1% of Tween20, protect this solution from the light.