GST pull-down assay

Reagents:

1. Sepharose-4B Glutathione (50 % vol/vol, GE Healthcare #17-0756-05);

2. 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES, Sigma-Aldrich #H3375);

3. Potassium Chloride (KCl, #P9333);

4. Glycerol (AR >99.0%, Sinoreagent #10010618);

5. Acrylamide (CP >98.0%, Sinoreagent #80001326);

6. N,N'-Methylenebisacrylamide (CP >98.0%, Sinoreagent #30117826);

7. Tris-glycine electrophoresis running buffer (10×, Bio-Rad #161-0734);

8. 2× Laemmli sample buffer (Bio-Rad #161-0737);

9. Bio-Safe Coomassie Stain (Bio-Rad #161-0786);

10. Potassium hydroxide (KOH, Sigma-Aldrich #221473);

11. Tris base (Sigma-Aldrich #T1503);

12. Ammonium persulfate (Sigma-Aldrich #A3678);

13. N,N,N′,N′-Tetramethylethylenediamine (TEMED, Sigma-Aldrich #T9281).

Instruments:

1. Mini-PROTEAN® Tetra Cell (Bio-Rad #165-8000);

2. PowerPac™ Basic Power Supply (Bio-Rad #1645050);

3. Centrifuge 5424 R (Eppenforf);

4. FluorChem FC3 System (Proteinsimple).

Consumables:

1. 50 ml centrifuge tube (Corning);

2. 500 ml Flask (Fisher);

3. 500 ml amber reagent bottle (Customization)

Reagents setup:

1. Prepare Acr-Bis (29:1 % wt/vol) solution:

       Take 145 g acrylamide and 5 g N,N'-Methylenebisacrylamide, dissolve into deionized water to a final volume of 500 ml, store in 500 ml amber reagent bottle in the dark at 4 °C.

2. HBS7.6 buffer formula:

              HEPES   5.96 g

              KCl        11.18 g

              Glycerol  100 ml

       Add deionized water to a final volume of 1 L, adjust the pH to 7.6 with KOH.

3. 12 % poly-acrylamide gel formula (2 gels):

Separating gel (12%, 10 mL):

               2 ml ddH2O

               4 ml 30 % Acry-Bis (29:1 % wt/vol)

               3.75 ml 1M Tris-Cl pH 8.4

               100 ul Ammonium persulfate (10 % wt/vol)

               5 ul TEMED

Stacking gel (4%, 5 mL):

    3.4 ml ddH2O

    0.83 ml 30% Acry-Bis (29:1 % wt/vol)

    0.63 mL 1M Tris-Cl pH 6.8

    50 ul Ammonium persulfate (10 % wt/vol)

    5 ul TEMED

Experimental setup:

1. Pre-incubation of the SNARE complex:

       Incubate the SNARE complex overnight at 4 °C. Take 10 ul sample, add 10 ul 2× Laemmli sample buffer. Divide the sample into two tubes, one is boiled at 100 °C for 10 min, the other does not handle. Analyze the two samples with SDS-PAGE at 35 mA steady current, followed by staining with Bio-Safe Coomassie Stain.

(NOTE: if the SNARE complex is assembled properly, an about 60 kDa band will be seen on the poly-acrylamide gel. If the sample is boiled before running electrophoresis, then the band will not be seen.)

2. Prepare pull-down samples:

       Incubate the samples at 4 °C for 2 hours with gentle shaking. After incubation, the media were washed by HBS7.6 buffer 3 times (1 ml for each time) using Centrifuge 5424 R at 500 ×g, 4 °C. The samples were left to a final volume of 50 ul, add 50 ul 2× Laemmli sample buffer. Analyze the two samples with SDS-PAGE at 35 mA steady current, followed by staining with Bio-Safe Coomassie Stain. The gels were captured using FluorChem FC3 System.

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