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Western blot

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A. Preparation of the sample:

1. Take 20-35 ug of your protein extract.

2. Add the appropriate amount of 4x sample loading buffer.

3. Sonicate for 5’ and boil the sample at 95-100 degrees for 5’

B. Electrophoresis:

1. Assemble gel in gel tank

2. Load carefully the selected amount of protein on each lane.

3. Keep one lane to load a protein MW marker.

4. Run at 100V-150V(constant voltage) until the blue front reaches the bottom.

E. Antibody detection:

1. Incubate with primary antibody diluted in 5% albumin in PBS overnight at 4 degrees.
You must follow manufacturer indications for the appropriate dilution.

2. Wash blot 3x 10 min with PBS.

3. Incubate the membrane with secondary antibody (HRP-labeled) at he appropriate dilution in 5% milk in PBS for 1h.

4. Wash blot 3x 10 min with PBS.

5. Incubate the membrane in 5 ml ECL.

6. Dry the membrane with care and place it between two transparent films.

7. In a dark room, place the mambrane inside the cassete and expose the photographic film for the appropriate time.

                         Detection of troponin T on porcine cardiac tissue lisates

G. Solutions/reagents

1. Gels: To calculate the appropriate amount of reagents for each percentage of acrylamide click here.

2. 4x SDS sample buffer (0.25M Tris-HCl pH 6.8, 8% SDS, 30% Glycerol, 0.02% Bromophenol Blue) containing a reducing agent (either 10% B-Mercaptoethanol or 0.3M DTT)

3. 1 x SDS Running Buffer made from 10x stock (30.3g tris, 144g Glycine, 10g SDS and make up to 1 L with water)

4. Transfer buffer (25mM Tris pH8.5, 0.2M Glycine, 20% Methanol).

5. PBS: 1X PBS + 0.1% Tween.

6. Ponceau S solution (0.1% ponceau, 5% acetic acid)

7. Stripping buffer: 62.5 mM Tris-HCl (pH 6.8), 2% SDS. (add 0.1 M 2-Mecaptoethanol).

C. Preparation of the membrane for the blotting step:

1. Cut a piece of nitrocellulose membrane.

2. Cut 10-12 pieces of filter paper of the same size.

D. Western blotting membrane Transfer:

1. Prewet the sponge, filter papers, gel and membrane.

2. Assemble the sandwich into the transblot: Black (negative charge)-sponge, filter paper (x5)-gel-membrane-filter paper (x5)-sponge-red (positive charge).

3. Transfer at 200-250 mA(around 100V) for 1-2 hour with or pre-chilled buffer. Take into account that larger proteins will take longer to transfer.

4. Take out the membrane and give it brief washes with ddH2O.

5. Check the presence of protein using Ponceau red.

6. Add in 5 % milk in PBS and block the membrane for 30’ at RT.

F. Western blot stripping protocol:

1. Rinse blot with 0.1% Tween 20 in PBS.

2. Add Strpping buffer 37 C 30 min

3. Rinse blot with 0.1% Tween 20 in TBS.
4. Start again with membrane blocking step and re-probe with new antibody.

cardio research,
17 Jan 2011, 03:20