General Guidelines for Preparing Samples for MS Analysis



Guidelines for preparing in-gel samples for MS Analysis

Gel Excision and Storage:

First of all, we recommend loading from 100 to 500 ng of the unstained Bio-Rad standards as MW markers (cat. nb. 161-0317). This will give you (and us) a rough idea on the amount of proteins there is in each band. Leave at least one empty lane between each condition/sample.

Wear a clean lab coat, Nitrile gloves, hat and full face when handling the gel (especially when excising the bands/spots). Use cleaned tubes and glassware (2-3 washes with HPLC grade 50%ACN/water) and cleaned pipet tips (2-3 washes with the working solution/buffer).

Excise bands/spots in a clean room (if available) or on a clean bench away from walking around spaces. NEVER WORK IN A CHEMICAL FUME HOOD.

When staining is complete, obtain an image as soon as possible. Please provide us with this image when you submit the samples. Cover the gel/container with food wrap as much as possible (i.e. do not expose the gel to air for too long).

Long term gel storage is not recommended. Protein oxidation and degradation occur with time, as well as an overall loss in signal quality.

We prefer the gel wet and maintained in water if ready to perform band excision. Otherwise, you can maintain the gel in a distaining solution for few days at 4ºC:

50:10:40 Methanol:Acetic acid:Water

30:7:63 Methanol:Acetic acid:Water

30:7:63 Ethanol:Acetic acid:Water

N.B. Soak the gel in water for 30 minutes before excising the gel bands/spots

As soon as possible, choose and excise the gel bands/spots on a clean plate or in the distaining vessel, with a clean scalpel and tweezers. Place the gel slice in a pre-cleaned sample tube of 1.5mL (we suggest to wash it twice with HPLC grade H2O) and add 10-20 uL of high quality water. Cut the gel bands/spots as close to the stained region as possible but keep the following dimensions:

Gel thickness: 1 mm (ideal for us) For 2D gel spots:

Height: from 2 to 5 mm minimum: 3 by 3 mm

Width: from 5 to 10 mm maximum: 6 by 6 mm

Gel thickness: 1.5 mm

height: from 1 to 3 mm

Width: from 5 to 7 mm

N.B. Smaller bands might be rejected and larger bands will be split into several samples and extra fees will be applied.

Do not store the gel in glycerol − contamination.

Do not dry the gel onto filter paper − contamination.

Avoid drying the gel between clear films − contamination.

Store the gel bands/spots at 4 oC (if less than a day) or at -80 oC.

Gel Visualization Methods

Use fresh solutions/buffers for all steps (or solutions/buffers dedicated to proteomics' samples). Wear a clean lab coat, gloves, hat and face mask when handling the gel (especially when excising the bands/spots). Use cleaned glassware (2-3 washes with HPLC grade 50%ACN/water) and cleaned pipet tips (2-3 washes with the working solution/buffer).

Cover the gel container with food wrap as much as possible (i.e. do not expose the gel to air for too long). USE A GLASS CONTAINER.

Silver Stain:

LOD (visual) ~1-10 ng (not quantitative method)

USE OUR SILVER STAINING PROTOCOL, it’s cheap and efficient (see below).

N.B. - Try to avoid over staining (bands that appear with over staining might not be identifiable by LC-MS/MS). Try to increase the amount of proteins instead.

- This distaining protocol causes artifacts (sulphonation/sulfation) for the identification of phosphorylated sites. Use Coomassie, colloidal Coomassie or fluorescent staining instead.

Coomassie Brilliant Blue (R-250):

Inexpensive, semi-quantitative, simple

LOD~ 60-100 ng.

Cover gel with staining solution and incubate from 0.5 to 4 hr/decant. (0.1% CBB R-250, 50%

methanol, 10% acetic acid). Briefly rinse gel with analytical water to remove residual stain/decant.

Distain gel in 30-50% methanol: 5-10 % acetic acid: analytical water/decant

30 minutes soaking in water before excising the bands

Colloidal Coomassie (G-250):

LOD ~2-10 ng.

Wash gel 3´ (10-15 min) in analytical water to dilute SDS/decant.

Just cover gel with Colloidal Coomassie stain (~4-6 hr)/decant:

Rinse 2´ with analytical water (1 min)/decant.

Distain gel with analytical grade water until background is low (~4 hours).

30 minutes soaking in water before excising the bands

Fluorescent Methods:

Sypro Ruby/Red/Orange/Tangerine, Ruthenium II, Deep Purple

Recommended for both visualization and MS

Very sensitive, quantitative (orders of magnitude), simple, more expensive, required UV plate for visualization and band excision.

LOD ~1-10 ng, preferred method for low abundance proteins

Follow the manufacturer protocol.

Caution: Photobleaching occurs with a “half life” of ~15 min in transiluminators.

30 minutes soaking in water before excising the bands

Silver staining for LC/MS/MS analysis

✓ Use a clean glass container to stain the gel and do not touch the gel with your hands. A large gel will require 250 to 500 ml of each solution. All washes and rinses should be performed while gently rocking the glass container.

1. Fix the gel in 50% methanol: 10% acetic acid for 30 min. Repeat once at least 2h to O/N.

✓ 125 ml methanol + 25 ml acetic acid + 100 ml H20

2. Rinse the gel with 20% ethanol for 20 min.

✓ 50 ml ethanol + 200 ml H20

3. Rinse the gel in water for 20 min.

4. Reduce the gel with sodium thiosulfate (0.2 g/L) for 2 min.

✓ 0.05 g sodium thiosulfate + 250 ml H20

N.B. Keep 25 ml for step 8 (for the developing solution).

5. Rinse twice 20 sec with water.

6. Incubate in silver nitrate (2 g/L) for 30 min.

✓ 0.5 g silver nitrate + 250 ml H20

7. Rinse once with water for 20 sec.

8. Wash the gel once with the developing solution (about 100-200 ml), discard the wash and develop to desired intensity with the remainder of the developing solution.

Developing solution contains sodium carbonate (30 g/L), formaldehyde (1.4 ml of a 37% solution/L), and sodium thiosulfate (10 mg/L, i.e. 25 ml of the previous solution, step 4, for 500 ml).

✓ 15 g sodium carbonate + 475 ml H20

✓ 25 ml of sodium thiosulfate (0.2 g/L) – step 4

✓ 700 (l of formaldehyde

9. Stop the reaction by exchanging the developing solution with 1% (v/v) acetic acid. Incubate for a minimum of 30 min.

✓ 2,5 ml acetic acid + 250 ml H20

IMPORTANT: try to avoid developing the gel more than 2-3 minutes since over staining reduces sensitivity for proteomic analysis (developing time must be recorded).

Store at – 80oC, the bands can be stored frozen for months.

Products

Sodium thiosulfate S446-500 Fisher

Silver nitrate S181-25 Fisher

Sodium carbonate S263-3 Fisher

Formaldehyde F79-4 Fisher

Acetic acid, glacial A38 212 Fisher

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