Protocol

Preparation and Loading of Protein Samples for Microinjection

This protocol was adapted from "Microinjection of Fluorophore-Labeled Proteins," Chapter 5, in Live Cell Imaging (eds. Goldman and Spector). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

This protocol provides methods for the preparation of protein samples and for loading them into pulled microinjection pipettes. Stock solutions of proteins are thawed, diluted (if desired), centrifuged at high speed to remove aggregates, and kept on ice until loading. Loading into micropipettes can be done using either a "front-loading" or a "backfilling" procedure; both methods are described below.

RELATED INFORMATION

Following pipette loading, microinjection is performed as described in Microinjection of Protein Samples.

MATERIALS

Reagents

Microinjection buffer A (optional; see "Sample Preparation," Step 4)

Microinjection buffer B (optional; see "Sample Preparation," Step 4)

Protein sample to be microinjected

Stocks are typically stored in 5-10 µl aliquots (10 µg/µl) in liquid nitrogen or at -80°C.

Proteins tagged with HA (hemagglutinin epitope) or GST (glutathione-S-transferase) tend to aggregate. To avoid frequent clogging of the micropipette tip, the tag should be cleaved prior to microinjection.

Equipment

Forceps

Hamilton microsyringe, 10 µl, or special loading plastic tips (Eppendorf)

These are used for loading micropipette tips via "backfilling."

Micropipette holder

Pulled micropipettes, prepared following puller manufacturer’s instructions

A tip diameter of ~0.3 µm is optimal.

Pulled micropipettes should be used within 1 week.

If desired, micropipettes can be sterilized or silanized inside a multipurpose pipette storage container. Pipettes are silanized by adding silane to the reagent well in the center of container. A container with pipettes in situ can be dry-heat sterilized at 170°C.

Ultracentrifuge with appropriate rotor (e.g., a TLA-100 rotor).

If an ultracentrifuge is not available, a microcentrifuge may be used (see Step 9).

METHOD

Sample Preparation

1. Cool the ultracentrifuge to 4°C.

2. Remove an aliquot of the protein from the freezer and place it on ice.

3. Allow the sample to melt.

4. If dilution is needed, dilute the sample with microinjection buffer A or microinjection buffer B.

5. Transfer the sample to a centrifuge tube appropriate for the intended rotor.

6. Insert the tube into a precooled (4°C) rotor.

7. Balance the sample with an additional tube containing an equal volume of liquid.

8. Insert the rotor into the centrifuge.

9. Centrifuge at 200,000g for 20 minutes at 4°C (e.g., using a TLA-100 rotor at 50,000 rpm).
Alternatively, the sample can be centrifuged in a microcentrifuge at maximum speed for no longer than 5-10 minutes (to avoid warming of the sample).

10. After centrifugation, remove the sample tube from the rotor and place it on ice.
Refreezing the sample prior to microinjection is not recommended.

Loading Micropipettes with Sample

11. Fill the micropipette using one of the two methods given below.
Micropipette loading from the tip ("front loading"):

i. Attach the micropipette to the holder/instrument collar that is connected to the gas pressure controller with rubber tubing.

ii. Remove the tube containing the sample from the ice bucket.

iii. Place the tip of the micropipette into the top of the solution (sample is drawn into the micropipette by capillary action).

iv. Press the "fill" pushbutton on the gas pressure controller to apply suction of about 82 kPa. The suction is applied as long as the button is left in.

v. When a sufficient amount of solution is drawn into the tip of the micropipette, release the "fill" pushbutton.

vi. Return the sample to ice.

Loading of the micropipette from the unpulled end ("backfilling"):

i. Precool the microsyringe by placing it on the ice.

ii. Load the microsyringe with 2-3 µl of sample (take care not to disturb the pellet) and place it back on ice.

iii. Use forceps to remove a micropipette from the storage container.

iv. With the other hand, insert the needle of the syringe into the barrel of the micropipette.

v. Inject a small amount of the sample (0.1 µl) into micropipette.
The tip of a micropipette fabricated from tubing with inner filaments will fill quickly. Discard any micropipette that is not filled to the very tip.

vi. Return the syringe to the ice.

vii. Attach the micropipette to the holder.

12. Continue with microinjection as described in Step 11 in Microinjection of Protein Samples.