Western Blotting with Alkaline Phosphatase Conjugates

SAMPLE PREPARATION

For Protein Concentration Determination of Cell Culture

1.Decant medium from 10cm dish of adherent cells and rinse plate rapidly with phosphate-buffered saline (PBS).

2.Aspirate excess PBS.

3.Add 1ml boiling lysis buffer (1% SDS, 1.0mM sodium ortho-vanadate, 10mM Tris pH 7.4).

4.Scrape cells from dish, transfer to a microcentrifuge tube, and boil for an additional 5 minutes. To reduce viscosity, the sample may be sonicated briefly or passed several times through a 26-gauge needle.

5.Centrifuge the sample for 5 minutes to pellet insoluble material, then discard pellet.

6.Dilute an aliquot of the cell lysate sample at least 10-fold for the BCA (Pierce) protein concentration assay (SDS concentration must be below 0.1% to avoid interference with the colorimetric reading).

For Protein Gel Electrophoresis of Cell Culture (without determining protein concentration)

1.Decant medium from 10cm dish of adherent cells and rinse plate rapidly with phosphate-buffered saline (PBS).

2.Aspirate excess PBS.

3.Add 1ml boiling 2X concentrated electrophoresis sample buffer (125mM Tris pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, 1.8% beta-mercaptoethanol).

4.Scrape cells from dish, transfer to a microcentrifuge tube, and boil for an additional 5 minutes. To reduce viscosity, the sample may be sonicated briefly or passed several times through a 26-gauge needle. Centrifuge the sample for 10 minutes to pellet insoluble material. Discard pellet.

5.The cell lysate sample (supernatant) is now ready for loading onto your gel.

For Protein Concentration Determination of Whole Tissue

1.Rapidly homogenize every 0.25g tissue in 3.5ml of boiling lysis buffer (1% SDS, 1.0mM sodium ortho-vanadate, 10mM Tris pH 7.4).

2.Microwave for 10-15 seconds.

3.Centrifuge the homogenate (16,000 x g, 15 C) for 5 minutes to pellet insoluble material, then discard pellet.

4.Dilute an aliquot of the tissue lysate sample at least 10-fold for the BCA (Pierce) protein concentration assay.

POLYACRYLAMIDE GEL ELECTROPHORESIS

Guidelines for choosing the percent gel to be used for certain molecular weight proteins (based on 37:1 acralyamide: bis acraylamide ratio)

4-5% gels: > 250 kDa

7.5% gels: 250-120 kDa

10% gels: 120-40 kDa

13% gels: 40-15 kDa

15% gels: < 20 kDa

Gel Electrophoresis

1.If not already in electrophoresis sample buffer, add an equal volume of 2X sample buffer (125mM Tris pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, 1.8% b-mercaptoethanol) to all samples and boil for 3-5 minutes.

2.Apply 5-20μg total protein of cell or tissue lysate to each well of a 0.75-1.0mm thick gel. For thicker gels (1.5mm thick), apply up to 25-40μg in each well.

3.Electrophorese until the bromophenol blue in the samples reaches the bottom of the gel. Turn off power supply. Keep gels in running buffer until ready to transfer.