Processing of Sdspage and Radiography

8. Transfer gel to 300 mL of fixing solution in a glass container.

9. Place the container onto a shaker and shake at 62.5 rpm for 30 min.

10. Drain solution and replace with a fresh 300 mL of fixing solution and shake at 62.5 rpm for 30 min.

11. Transfer gel to a square Petri dish.

12. Add 15 mL of Entensify Enhancer Part A to dish.

13. Shake on shaker at 125 rpm for 45 min.

14. Add 15 mL of Entensify Enhancer Part B

15. Shake on shaker at 125 rpm for 45 min.

16. Dry gel in a Gel Dryer at 60°C for 120 min, until completely dry.

17. Place the dried gel on a radiogram cassette and expose an X-ray film to the gel in a dark room.

18. Place the cassette in a -80°C freezer for hours to days (depending on the intensity of radioactivity) (see Note 4).

19. Develop the film in an X-OMAT (Kodak) machine. Alternatively, bands in the gel can be quantified on an Instant Imager (Packard). The methionine-labeled (Pro-Mix) proteins are shown in Fig 2.

3.5. Characterization of Expressed Proteoglycans

1. Transfer 30 ^L of eluate (from Subheading 3.3.3., step 11) to a 1.5-mL boiling Eppendorf tube (For a 60-^L digest reaction).

Fig. 2. Methionine labeling experiment. (A) Spent medium: autoradiography of empty vector (control, lane 1) and aggrecan G3 construct (lane 2) from wild-type CHO cells (1). Note that the diffuse band in lane 2 shows a typical proteoglycan pattern (bracket). (B) Cell lysates: autoradiography of empty vector (lane 1) and aggrecan G1 construct (lane 2) from wild-type CHO cells (1). Note the intracellular G1 core protein (arrow, lane 2).

Fig. 2. Methionine labeling experiment. (A) Spent medium: autoradiography of empty vector (control, lane 1) and aggrecan G3 construct (lane 2) from wild-type CHO cells (1). Note that the diffuse band in lane 2 shows a typical proteoglycan pattern (bracket). (B) Cell lysates: autoradiography of empty vector (lane 1) and aggrecan G1 construct (lane 2) from wild-type CHO cells (1). Note the intracellular G1 core protein (arrow, lane 2).

Was this article helpful?

0 0
Healthy Chemistry For Optimal Health

Healthy Chemistry For Optimal Health

Thousands Have Used Chemicals To Improve Their Medical Condition. This Book Is one Of The Most Valuable Resources In The World When It Comes To Chemicals. Not All Chemicals Are Harmful For Your Body – Find Out Those That Helps To Maintain Your Health.

Get My Free Ebook


Post a comment