×
Please leave your comments or questions
 

In-gel digestion for mass spec

Author: Tie-Mei Li, updated date: , view: 440, Q&A: 0

Procedure

Stain protein gel: For silver staining, follow the basic stain protocol in SilverQuest™ Silver Staining Kit (LC6070). For coomassie staining, avoid methanol in all buffers when the purpose is to identify methylation sites.

Cut gel: cut the target gel piece into 1 mm3 pieces on a clean glass plate of plastic sheet. Put the gel pieces in a 1.5 mL tube.

Destaining after silver stain: Follow the destaining procotol described in SilverQuest™ Silver Staining Kit.  

Destaining after coomassia stain: destain the gel pieces with 1mL 50% acetonitrile at 37°C,  10min x 3 times or until no blue color. Wash with 1mL water for 10min x 3 times.

In-gel Digestion:

1. Destain the gel and wash thoroughly with water as described above.

2. Add 1 mL DTT (10 mM), 56 °C for 40 min.

3. Remove DTT, add 50 mM IAA to cover the gel slices (make 500 mM stock fresh each time: dissolve 46.4 mg Iodoacetamide in 0.5 mL 50 mM NH4HCO3 ). React at room temperature for 1 hr in the dark.

4. Wash twice with 1 mL water, 20 min each time.

5. Add 1 mL acetonitrile to make the gel shrink. Mix by turning the tube upside down for several times. Remove the acetonitrile and repeat once (until the gel turn white).

6. Speedvac for 5 min to remove the remaining acetonitrile.

7. Prepare 10 ng/uL trypsin in 50 mM NH4HCO3. Add 100-200 ng trypsin to each sample (usually make trypsin:protein = 1:20 – 1:50 by weight, if don’t know the amount of the proteins, use 200 ng trypsin)

8. Incubate at 37 °C for 15 min. Check if the liquid can cover the gel slides. If not, add 50 mM NH4HCO3 until the buffer cover the gel slides. Digest at 37 °C overnight.

9. Add 30-50 uL Extraction Buffer I (50% acetonitrile, 1% formic acid in water) to extract peptides from the gel. Rotate at room temperature for 30-60 min. Transfer the liquid to a fresh tube.

10. Add 30-50 uL Extraction Buffer II (75% acetonitrile, 1% formic acid in water) to the gel slides and rotate for 30-60 min. Combine the liquid with the one from step 9.

11. Specdvac to remove acetonitrile (or dry the sample for long-term storage). 

12. Continue to Stagetip.

 

Stage tip

  1. prepare      C18 stagetips (we use the Thermo Scientific SP301)

  2.  load stage tips with:1x 50uL MeOH, spin      at 3,000 rpm for 1 min (use this speed in the following steps unless noted      otherwise)*

  3. 50      uL Buffer B (= elution buffer)*

  4. 2x      50 uL Buffer A(=wash buffer)*

  5. Acidify      samples with 5 uL 10% formic acid, or until pH<3.

  6. Load      samples on stage dips

Spin at 2,000 rpm, 5 min (slower speed so the peptides can bind to C18)*

  1. Wash      1x 50uL buffer A*

*important note: make sure all liquid is gone before you load next step

  1. Elute      with 30 uL Buffer B , spin at 2,000 rpm, 5 min (slower speed to elute      peptides)

  2. Speedvac      plate until dry, store at -20 degree

 

For LC-MS:

  1. Resuspend      the peptides in 10 uL (2% acetonitrile, 0.1% formic acid in water), vortex

  2. Spin      10 min at max speed to remove any pellet.

  3. Transfer      to sample vial. Send to Elias lab.

 

Buffer A: 0.1% formic acid in water

Buffer B: 60% acetonitrile, 0.1% formic acid in water


Comment
Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.