Protocol by PKU 2010

         Notebook > Protocols > SDS-PAGE & Western Blot protocol

SDS-PAGE Protocol

1. Selection of a SDS-PAGE gel. Typically 10% acrylamide gels are used for high molecular weight (MW) proteins (>50 kDa), 12% gels for mid range MW proteins (15 - 50 kDa), and 15% gels for low MW proteins (<15 kDa). Load 5 ul marker or 20 ul protein sample each lane.

2. Run at 80V until samples enter the separation gel in gel running buffer (19.3 mM Glycine, 2.5 mM Tris base, 0.1% SDS), and then run at 120V. Electrophoresis is complete when the dye front migrates about 2 mm from the bottom of the gel..

3. Stain with Coomassie brilliant blue for 1 h, and then destain in destain buffer (50% H2O, 20% AcOH, 30% methanol) for 1h.

Western Blot Protocol

1. After loading 5 ul prestained protein marker or 20 ul His-Tag fusion protein sample each lane, run SDS-PAGE (without staining).

2. Cut a piece of PVDF membrane and wet in 10 ml methanol for 5 min. Then add 40 ml distilled water and shake for 10 min. Transfer PVDF membrane to western transfer buffer (39 mM glycine, 48 mM Tris base, 0.037% SDS, 20% methanol)untill use.

3. Balance the gel and two holders in western transfer buffer for 10 min.

4. Assemble "sanwich":

Black (negative charge)-holder-gel-membrane-holder-red (positive charge)

5. Transfer at 20 mA/lane for 20 min.

6. Wash membrane for 10 min, with 15 ml 1X TBS (150 mM NaCl, 20 mM Tris-HCl pH7.5)

7. Incubate for 1 h in 27 ml blocking buffer (dissolve 5% skimmed milk powder in TTBS (dissolve 0.1% Tween20 in TBS)).

8. Wash three times, each for 10 min, with 20 ml TTBS.

9. Incubate for 1 h in His-Tag Monoclonal Antibody diluted 1:800 in blocking buffer.

10. Wash three times, each for 10 min, with 20 ml TTBS.

11. Incubate for 1 h with Rabbit anti-Mouse IgG AP Conjugate diluted 1:800 in blocking buffer.

12. Wash three times, each for 10 min, with 20 ml TTBS.

13. Prepare developing solution by adding 60 μl NBT solution and 60 μl BCIP solution to 15 ml AP buffer.

14. Place membrane in clean tray and add developing solution. Incubate membrane at room temperature until color develops. Strong purple signals should appear within 2–10 min.

15. To stop reaction, wash blot thoroughly in deionized water. Allow to air dry. Store dry blots at room temperature wrapped in plastic.

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