Presupuesto para la ejecución del Plan Comunicación

and

Immunoblotting

15. Carefully overlay the samples in each well with the BN cathode buffer. Fill the upper/inner chamber with BN cathode buffer (containing 0.02% w/v Coomassie blue as described in Reagents and Solutions) and the lower/outer chamber with BN anode buffer.

16. Connect electrodes. If a small gel has been prepared (e.g., using BioRad Protean II or III), run at 100 V; if a large gel has been prepared (e.g., using BioRad Protean II xi), run at 150 V. Continue electrophoresis at the appropriate abovementioned voltage until the sample has entered the separating gel. At that point, increase voltage to 180 V for small gel or 400 V for large gel.

Coomassie blue comes into contact with the samples inside the wells during the electro- phoresis.

17. Remove the BN cathode buffer (especially from within the wells) after two-thirds of the gel run. Fill the upper/inner chamber with BN cathode buffer (low Coomassie blue) that contains only 0.002% (w/v) Coomassie blue. Continue electrophoresis at 180 V for small gel or 400 V for large gel.

This procedure ensures that the individual lanes can be identified after the gel is run and can be omitted if a second-dimension gel is not to be applied (see, e.g., Alternate Protocols 1 and 2), or if precipitated material is visible between the stacking and separating gel. 18. Turn off power supply and remove the electrodes once the dye front has reached the

bottom of the gel.

Extrude and store Blue Native gels

19. Remove the glass plates, with the gel in-between, from the electrophoresis apparatus. 20. Open the glass plates by lifting the smaller one and keeping the gel attached to the

bigger, bottom plate. Remove the stacking gel with the smaller glass plate.

21. Cut away the lanes where the marker mixes were loaded from the rest of the gel using the smaller glass plate. Visualize the marker proteins by standard Coomassie blue or silver staining (UNIT 6.6).

For some purposes, the ferritin marker (440 and 880 kDa), which is visible without staining, is sufficient. When assigning the positions of the marker, take into consideration that the gel might shrink upon staining.

Alternatively, after the first-dimension BN-PAGE, proteins can be stained directly with silver or Coomassie brilliant blue (UNIT 6.6), or detected by immunoblotting (western blotting; Alternate Protocols 1 and 2). Optionally, stained spots can be cut out and the proteins identified by mass spectroscopy.

22. Stamp out each individual lane with the smaller glass plate. Either immediately run the second dimension SDS-PAGE (Basic Protocol 2), or freeze each lane wrapped in aluminum foil at−20◦C (stable at least 1 year).

Do not bend a frozen gel piece, since it breaks easily. Alternatively, the first-dimension BN gel can be transferred to a membrane followed by immunoblotting to visualize the proteins of interest (Alternate Protocols 1 and 2).

SUPPORT PROTOCOL PREPARATION OF CELL LYSATES FOR BLUE NATIVE GEL

ELECTROPHORESIS

Blue Native (BN) gel electrophoresis is suitable for the separation of pure proteins as well as complex protein mixtures such as subcellular fractions or cell lysates. Sample preparation is one of the critical steps in performing high-quality BN gels. First, the proteins have to be present in soluble and native form. Thus, the choice of detergent is important and one should start BN experiments by testing several nonionic detergents

Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis

for the protein/protein complex of interest (see Commentary). The detergent should be effective enough to extract the proteins from cellular membranes, but at the same time mild enough to keep multiprotein complexes intact. The most commonly used detergents are given in Reagents and Solutions. The proteins must be eluted in native form, if the purification procedure includes binding to a matrix, as e.g., in immuno- or affinity purifications (often referred to as immunoprecipitation; seeUNIT 7.2). Second, the sample

has to be prepared without potassium or divalent cations, since Coomassie blue and Coomassie blue–bound proteins precipitate with those ions and consequently do not enter the gel. Hence, cations that interact with Coomassie blue have to be removed and substituted by 6-aminohexanoic acid, in order to maintain a certain ionic strength of the solution, which is necessary for the solubility and stabilization of many protein complexes. Sodium ions are tolerated to a maximum concentration of 50 mM. Third, samples have to be loaded with detergent on BN gels. Otherwise, proteins aggregate during the stacking step of the electrophoresis.

Membrane and organelle fractions lysed in BN lysis buffer can be directly applied to BN gels. Cellular lysates have to be dialysed against BN dialysis buffer, in order to remove small cations and metabolites. Proteins bound to a matrix are washed and eluted in BN dialysis buffer.

This protocol describes the preparation of cell lysates. Cells are washed and lysed in any lysis buffer, although we recommend using the BN lysis buffer. After removal of insoluble material, the lysate is dialysed against BN dialysis buffer. In this protocol, a self-made dialysis setup utilizing dialysis membranes is described. Alternative desalting methods can be applied as well.

Materials

Cell culture dish (10 to 15 cm) containing cells of interest (80% confluent) Phosphate-buffered saline (PBS; see recipe), ice cold

BN lysis buffer (see recipe) BN dialysis buffer (see recipe) Cell scrapers

10- to 50-ml centrifuge tubes

Refrigerated cell culture centrifuge (with adaptor cavities for microcentrifuge tubes in rotor accommodating 50-ml tube)

Dialysis membranes, MWCO 10 to 50 kDa (boiled and kept at 4◦C in 0.001 M EDTA)

1-ml reaction tubes (e.g., microcentrifuge tubes) Beaker (100-ml to 1-liter, depending on sample size) Harvest and wash the cells

1. Place cell culture dish containing cells on ice.

2. For adherent cells, wash cells three times, each time with 2 to 10 ml ice-cold 1× PBS.

Cells grown in suspension are washed three times with ice-cold 1×PBS by repeatedly pelleting the cells by centrifugation for 5 min at 350×g, 4◦C.

The volume of PBS used for one washing step should be equal to the volume of medium in which the cells were cultivated.

3. Add 2 to 10 ml (i.e., volume in which cells were cultivated) of ice-cold 1×PBS to the dish, remove the cells using a cell scraper, and transfer the cell suspension to a 10- to 50-ml centrifuge tube.

Electrophoresis and

Immunoblotting

4. Collect cells by centrifugation for 5 min at 350×g, 4◦C, and remove supernatant. 5. Resuspend cells in 1 ml cold 1×PBS, transfer cell suspension into a small reaction

tube (e.g., microcentrifuge tube), and pellet cells by centrifugation for 5 min at 350 ×g, 4◦C.

6. Remove and discard the supernatant using a pipet.

7. Either move on to step 8 or freeze the cell pellet at−20◦C. Frozen cells can be stored at−20◦C for at least 6 months. Prepare the cell lysates

8. Resuspend the cell pellet in ice-cold BN lysis buffer (or other lysis buffer that has been used successfully to extract your multiprotein complex of interest).

The concentration of cells per volume lysis buffer depends on the amount of protein that should be loaded on the BN gel. As an approximation, use 106cells per 250 to 500 µl lysis buffer.

9. Incubate on ice for 15 min to allow cell lysis.

The lysis time can be prolonged, if it is convenient—e.g., in the case where many different samples are processed and some of them have to wait for the last ones.

10. Microcentrifuge 15 min at 13,000×g, 4◦C, to remove insoluble material. Prepare to dialyze the lysate

11. Melt a hole in the cap of a microcentrifuge tube using the large-diameter end of a hot Pasteur pipet. Chill the tube on ice.

12. Transfer the supernatant from step 10 into the chilled tube from step 11. 13. Place a dialysis membrane over the opened tube and close the cap.

Make sure that there are no folds or tears in the dialysis membrane. 14. Seal the cap at the edges with Parafilm.

Make sure that the hole in the tube is not covered by the Parafilm.

15. Invert the tube and centrifuge upside-down at the lowest speed possible in the adaptor cavity for 50-ml tubes in a cell culture centrifuge for 10 sec at 4◦C.

16. Prepare a 100-ml beaker with ice-cold BN dialysis buffer and a magnetic stirrer. Use at least 10 ml dialysis buffer per 100µl sample.

17. Fix the tube upside down inside the beaker and remove air bubbles from the hole beneath the cap.

Make sure that the dialysis membrane is not damaged. Dialyze lysate

18. Switch on the magnetic stirrer and dialyze for 6 hr or overnight in a cold room. Make sure that stirring is not creating air bubbles at the dialysis membrane.

19. Collect the dialysed cell lysate in a new chilled microcentrifuge tube and analyze by BN-PAGE (Basic Protocol 1).

If the sample will be subjected to two-dimensional analysis, reserve one-third of the sample to serve as a control in the second-dimension analysis.

Freezing and thawing of cell lysates might lead to the aggregation of proteins. Therefore, the cell lysate should be separated immediately by BN-PAGE. For some proteins, freezing might be possible and has to be determined empirically.

Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis BASIC PROTOCOL 2