Abstract: This protocol was used for tissues in detail to detect signals of protein

Preparation of Sample

1. Protein extraction

-Preparation of Tissue: 10-20 mg

-Homogenize sample in 0.6 ml PRO-PREP solution by using homogenizer.

-Incubate at –20⁰C for overnight.

-Centrifuge at 14.000 rpm for 10 mins at 4⁰C and transfer supernatant to a fresh tube (1.5 ml tube).

2. Protein measurement

-Prepare sample solution: 50 ml

             2 ml Protein plus 48 ml DW (dilution factor: 2)

-Prepare 1X Pro-Measure solution: Dilute 1 part of 10X Pro-Measure solution by adding 9 parts of DW. Vortex sufficiently.

-Add 500 ml 1X Pro-Measure solution into 50 ml sample solution. Vortex sufficiently.

-Incubate for 2-5 mins at RT.

-Measure absorbance at 595 nm (500 ml).

-Adjust Protein concentration: 6 mg/ml.

-Re-measure.

Note:

-Blank: 50 ml DW plus 500 ml 1X Pro-Measure solution.

-Pro-Measure solution on glass cuvette surface can be removed by EtOH (or MtOH).

3. Adjustment of Protein concentrations: (For Western Blot: 20-60 mg) by using DW.

4. Denaturation of sample:

-Sample plus DW/ sample buffer=4/1

For example: Sample plus DW=10 ml/ sample buffer=2.5 ml.

-Denature at 85⁰C for 5 mins and chill on ice for 5 mins.

Preparation of the gel

                                    Resolving Gel                Stacking Gel

30% Acrylamide	3.3 ml	0.65 ml
Resolving Buffer	2.5 ml
Stacking Buffer		4.3 ml
DW	4.1 ml
10% APS	200 ml	50-60 ml

-Pour the resolving gel to about 1 cm below the well of the comb.

-Seal with 400 ml DW. This step can stop here and leave the gel for overnight.

-When the resolving gel has set (about 15-30 mins), pour off DW.

-Pour the stacking gel and insert the comb immediately.

-When the stacking gel has set (about 40 mins), remove the comb and wash the well with DW.

-Place the gel in the gel rig and immerse in running buffer.

Running gel:

-Load sample into the well.

-Run with constant current (80 Volt) for 65 mins (60-70 mins)

Preparation of Membrane

-Cut a piece of the membrane: 8.5 cm x 4.5 cm

-Filter paper: 9.5 cm x 6 cm

Membrane transfer

-Membrane should be wetted in Methanol and transfer into transfer buffer.

-Pre-wet the sponges, filter paper (slightly bigger than gel) in transfer buffer. And keep the gel, membrane and filter paper at 10⁰C for 10 mins before transfer.

Sponge – Filter paper – Gel – Membrane – Filter paper – Sponge

-Transfer for 3 hours at 300 mA or overnight at 30 mA

Blocking membrane

-Let membrane dry at RT about several hours. Membrane should be wetted in Methanol before blocking.

-Block membrane in 5% Skim Milk (0.25g skim milk in 5 ml PBS-T) for overnight with shacking at 80 rpm.

Antibody Incubation

-Wash the membrane 3 times with PBS-T for 10 mins/each time

-Incubate the membrane with primary antibodies diluted 1/500 in Blocking Buffer (10 ml/5 ml PBS-T containing 250 mg skim milk) for 1 hour at RT with shacking at 80 rpm.

-Wash the membrane 3 times with PBS-T for 10 mins/each time

-Incubate the membrane with secondary antibodies diluted 1/3000 in Blocking Buffer (1.75 ml/5 ml PBS-T containing 250 mg skim milk) for 1 hour at RT with shacking at 80 rpm.

-Wash the membrane 3 times with PBS-T for 10 mins/each time.

-Incubate the membrane with chromogen (the ratio: 1/1)

-Expose to X-ray films for 30 sec and 1 min.

Reagents and Chemicals

5X Sample Buffer: 10 ml

1M Tris-HCl (pH: 6.8)	0.6 ml (60 mM)
50% Glycerol	5 ml (25%) or 2.5 ml
10% SDS	2 ml (2%)
2-Mercaptoethanol	0.5 ml (14.4 mM)
1% Bromophenol Blue	1 ml (0.1%)
DW	0.9 ml or 3.4 ml

Filtration and storage at –20⁰C

PBS-T:

One table per 100 ml DW plus 50 ml Tween 20.

Skim Milk (or BSA)

250 mg skim milk per 5 ml PBS-T.