Assessment of SREBP Activation Using a Microsomal Vesicle Budding Assay

Materials and reagents

Biological materials

Note: The general microsomal budding assay is not dependent on the animal or diet used.

1. Human SREBP-1c transgenic rat

2. High carbohydrate diet (MP Biomedicals, catalog number: 960238)

   
   

1. HEPES (Sigma-Aldrich, catalog number: H3375)

2. D-Sorbitol (Sigma-Aldrich, catalog number: S6021)

3. Potassium acetate (Sigma-Aldrich, catalog number: P1190)

4. Magnesium acetate tetrahydrate (Sigma-Aldrich, catalog number: M5661)

5. 0.5 M EGTA-KOH, pH 8.0 (Fisher Scientific, catalog number: 50-997-744)

6. 1 M Tris-Cl, pH 6.8 (Fisher Scientific, catalog number: 50-843-263)

7. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D9779)

8. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: 75746)

9. Bromophenol blue (Sigma-Aldrich, catalog number: B0126)

10. Glycerol (Sigma-Aldrich, catalog number: G5516)

11. Trizma base (Sigma-Aldrich, catalog number: T1503)

12. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340)

13. 0.9% Sodium chloride irrigation (Saline) (Baxter, catalog number: BX-2F7124)

14. Isoflurane (Piramal Critical Care, catalog number: 33794-013-25)

15. Creatine phosphate (Millipore Sigma, catalog number: 10621714001)

16. ATP (Sigma-Aldrich, catalog number: G6419)

17. GTP (Sigma-Aldrich, catalog number: G8877)

18. Creatine kinase (Millipore Sigma, catalog number: 10127566001)

19. Anti-HA antibody (Cell Signaling, catalog number: 3724)

20. Anti-Calnexin antibody (Enzo, catalog number: ADI-SPA-860-F)

21. Anti-ERGIC-53 antibody (Abcam, catalog number: ab125006)

22. Goat anti-rabbit IgG, F(ab’)2 (Jackson Immuno, catalog number: 115-035-072)

23. Pierce^TM BCA Protein Assay kit (Thermo Fisher, catalog number: A65453)

24. SuperSignal West Pico chemiluminescent substrate (Thermo Fisher, catalog number: 23225)

    
    

1. Buffer 1 (see Recipes)

2. Buffer 2 (see Recipes)

3. 5× SDS-loading buffer (see Recipes)

4. 20× budding reaction supplement (see Recipes)

   
   

Recipes

pH values of buffers are all adjusted at room temperature.

1. Buffer 1

   Reagent	Final concentration	Quantity or Volume
   1 M HEPES-KOH, pH 7.2	10 mM	500 μL
   2 M Sorbitol	250 mM	6.25 mL
   1 M KOAc	10 mM	500 µL
   1 M Mg(OAc)2	1.5 mM	75 µL
   Total	n/a	Adjust pH to 7.2 with KOH, then add H2O to 50 mL

   Can be stored at 4 °C for up to two months.

   
   

2. Buffer 2

   Reagent	Final concentration	Volume
   1 M HEPES-KOH, pH 7.2	50 mM	2.5 mL
   2 M Sorbitol	250 mM	6.25 mL
   1 M KOAc	70 mM	3.5 mL
   1 M Mg(OAc)2	2.5 mM	125 µL
   0.5 M EGTA-KOH, pH 8.0	5 mM	500 µL
   Total	n/a	Adjust pH to 7.2 with KOH, then add H2O to 50 mL

   Can be stored at 4 °C for up to two months.

   
   

3. 5× SDS-loading buffer

   Reagent	Final concentration	Volume
   SDS	15%	15 g
   Bromophenol blue	0.02%	20 mg
   Glycerol	25%	25 mL
   1 M Tris-Cl, pH 6.8	0.15 M	15 mL
   Total	n/a	Add H2O to 100 mL

   Add 62.5 μL of β-mercaptoethanol for each 500 μL of loading buffer freshly before use.

   
   

4. 20× budding reaction supplement

   The following reagents should be freshly prepared individually. Prepare the 20× budding reaction supplement by mixing equal volumes of each reagent immediately before carrying out the budding reaction.

   Note: The molecular weight or specific activity of each reagent will be different depending on the source and lot number of the reagents. The quantity needs to be calculated based on the information from the vendor.

   Reagent	20× concentration	Quantity	Final volume in H2O
   Creatine phosphate	200 mM	26.2 mg	100 µL
   ATP	30 mM	6.61 mg	100 µL
   GTP	10 mM	2.09 mg	100 µL
   Creatine kinase	80 U/mL	1 mg	10 mL

   
   

Laboratory supplies

1. 18 G catheter (BD, catalog number: 382644)

2. Protein LoBind tubes (Eppendorf, catalog number: 022431081)

3. Polycarbonate centrifuge tubes (for AT3 rotor) (Beckman Coulter, catalog number: 343776)

4. Polycarbonate centrifuge tubes (for AT6 rotor) (Beckman Coulter, catalog number: 362305)

Equipment

1. Tabletop centrifuge (Eppendorf, model: Centrifuge 5424)

2. 37 °C incubator (ThermoMixer, Eppendorf, model: F1.5 was used in this experiment)

3. Ultracentrifuge (Thermo Scientific, model: Sorvall MX120)

4. S100-AT6 fixed angle rotor (Thermo Scientific, catalog number: 45588)

5. S120-AT3 fixed angle rotor (Thermo Scientific, catalog number: 45584)

6. Peristaltic pump (Amersham Biosciences, model: Pump P-1)

7. Dounce tissue homogenizer-2 mL (DWK life sciences, catalog number: 885300-002)

8. Dounce tissue homogenizer-7 mL (DWK life sciences, catalog number: 885300-007)

Procedure