Proteomics Resource

2D PAGE Experimental Procedures

Sample Preparation - General considerations
Follow these guidelines for your first samples that you analyze by 2D PAGE. You can always modify your protocols later:

  • Discuss your project idea/questions/protocols with us before commencing anything!
  • Keep sample preparation as simple as possible to avoid losses
  • Minimize proteolysis, perform all steps at as low a temperature as feasible
  • Use sample preparation buffers recommended by us
  • Avoid SDS and salt (or remove before isoelectric focusing)
  • Store samples at -80 ° C and drop them off on dry ice
  • Remove all particulate matters by ultra-centrifugation
  • Never heat the sample after adding urea to avoid protein carbamylation
  • Optimize procedures after the first samples are analyzed under our standard conditions
  • Look at the basic way to submit sample to us

Cell Disruption
Ideally sample preparation will completely solubilize and denature proteins in the sample. This step is achieved by disrupting cells or tissues in a strong denaturing solution (such as a buffer containing urea and/or thiourea). Gentle lysis methods include osmotic lysis, freeze-thaw lysis, detergent lysis, or enzymatic lysis. More vigorous methods include sonication, French pressure cells, bead beating, grinding in liquid nitrogen, and mechanical homogenization using Dounce or Potter homogenizers. For the various types of samples we anticipate to be submitted here, we recommend the following disruption methods. More specialized techniques can be discussed, if required.

 

Sample Origin

Disruption method

Bacteria

 Sonication in 3 to 5 pellet volumes of sample buffer

Yeast

 Bead lysis and resuspension in 5 pellet volumes of sample buffer

Tissue culture cells

 Lysis in sample buffer and sonicate

Solid tissues

 Mechanical homogenization

Parasites

 Inquire

Others, including bodily fluids, protein solutions already in buffers, partially purified or pre-fractioned lysates

 Inquire

 

Protection Against Proteolysis
Protease inhibitors are recommended to be included in the sample preparation buffer. We recommend the following: 

 

Protease Inhibitor

Effective against

1mM PMSF (make up in DMSO or ethanol)

 Serine Proteases, Some  Cysteine Proteases

 Aprotinin

 Many Serine Proteases

 Leupeptin

 Many Serine and Cysteine Proteases

 Pepstatin

 Aspartyl Proteases

 

Protein Concentration/Precipitation
For a sample to be submitted, overall protein amount can vary from as little as 50 μg up to several milligrams (with option to dilute). Optimum sample volume (includes sample and HPR focusing buffers) per small (11cm) IPG strip is 200μL, and for large strips (18 cm) is 350 μl. 

Samples that are dilute (such as purified protein complexes, pre-fractionated proteins, body fluids) can be concentrated in various ways. Many of these procedures also remove contaminants that can interfere with 2D analysis, such as salt, detergents, nucleic acids, and lipids. Various precipitation methods exist:

Recommended Method & Instructions

 

Limitations

 

TCA in acetone precipitation

- Difficulty with resolubilizing protein precipitate completely.
- Low pH solution: degradation or modification may occur.
- Losses due to incomplete precipitation.

TCA precipitation

- Difficulty with resolubilizing protein precipitate completely.
- Residual TCA must be removed by washing with acetone or ethanol.
- Low pH solution: degradation or modification may occur.
- Losses due to incomplete precipitation.

Ammonium sulphate precipitation 

- Losses possible, but recommended as prefractionation procedure of complex  mixtures.
- Residual ammonium sulphate must be removed by dialysis.

Filtration using commercial spin devices

Protein loss by absorption to the membrane.

 

Non-protein contaminants can interfere with 2D analysis and their removal is recommended whenever possible. Additional methods to remove contaminants include:

 

Contaminant

 

Reason for Removal

 

Procedure

 

Salts and other charge  carrying molecules

- Salt results in high conductivity and poor focussing,  thereby causing horizontal streaking on gels.
-Waler movement can occur, resulting in drying of the  IPG strip.

 - Dialysis (very effective)
 - Gel Filtration (losses)
 - Precipitation (see above)

Endogenous small ionic  molecules (nucleotides,  metabolites)

Often negatively charged, resulting in poor focussing.

 TCA/Acetone precipitation is the  method of choice

Ionic Detergents

Negatively charged SDS/protein complexes focus poorly

 - Dilute in non-ionic detergent  (CHAPS, Triton X-100) to  0.25% SDS final
 - Acetone precipitation at room  temperature (will reduce protein  recovery) 

Nucleic Acids

- Increase sample viscosity, clogging gel pores
- Can bind to proteins, preventing focussing
- Stain with silver

- Treat with nuclease (may  appear on 2D gel)
 - Ultracentrifugation, but may  also remove high molecular  weight protein complexes

 

Sample Buffers
To achieve well-focused first dimensions, proteins must be completely solubilized. This is achieved by a combination of urea and detergent. Thiourea improves the solubilization of membrane proteins. The sample buffer must not affect the protein PI or retain high conductivity. SDS should NOT be used as a detergent as it is charged and forms complexes with proteins. The final concentration of SDS should be below 0.25% for IEF.

Reducing agents are required to break disulphide bonds. DTT is commonly used, but alternatives like TCEP (tris 2-carboxyethyl phosphine) are used now. Carrier ampholytes enhance protein solubility by minimizing charge-charge interactions. While low temperatures generally inhibit proteolysis, samples should remain in sample buffer at room temperature for 1 hour in order to aid complete solubilization. Heating of the sample in the presence of detergent may increase solubilization, but should be performed in the absence of urea, which could lead to protein modifcation. Sonication generally helps speed solubilization.

To submit your sample to us, we recommend a simplified Urea/Thiourea buffer, as listed below. We will add the other agents necessary that are listed in FOCUSING BUFFERS below, required for actual IEF.

 UTC Buffer (to submit samples to HPR)

 7 M Urea

 2 M Thiourea

 2 % CHAPS

 

Focusing buffers (used by HPR)

CHAPS Buffer  

 7 M Urea

 2 M Thiourea

 2 % CHAPS

 2 % Ampholytes

 65 mM DTT

 Serdolit MB-1

 0.01 % Bromophenol Blue

Zwittergent Buffer

 7 M Urea

 2 M Thiourea

 2 % CHAPS

 0.8 % Ampholytes

 1 % Zwittergent

 65 mM DTT

 Serdolit MB-1

 0.01 % Bromophenol Blue

ASB-15 Buffer. This buffer can either be used alone or as a 1:1 mixture with Zwittergent Buffer. This mixture was shown to increase resolution of basic proteins.

 7 M Urea

 2 M Thiourea

 5 mM TCEP (replaces DTT, more effective in reducing disulphide bonds,  more resistant to air oxidation, stable at 4C for months)

 1 % ASB-15 (alternative zwitterionic detergent, readily soluble in high concentrations of urea)

 1 % Triton X-100

 0.5 % CHAPS

 2 % Ampholytes 

 Serdolit MB-1

 0.01 % Bromophenol Blue

****While we recommend one of these buffers for initial analysis, we are always willing to work with individual investigators to optimize conditions.

Staining Protocols

Our facility currently provides the following:

  • Silver Staining (visible)
  • Coomassie Brilliant Blue (G-250) Staining (recommended, visible)
  • SYPRO Ruby Staining (fluorescent)
  • Flamingo Red Staining (recommended, fluorescent)
  • Pro-Q Diamond Staining (for phosphoproteins) (fluorescent)