Protein expression and purification

Preparation of colony blots

Chemiluminescent substrates are not recommended for use with colony blots.

Materials

Prepare the buffers, reagents, and agar plates. See tables 10% SDS, Denaturing solution, Neutralization solution, and 20x SSC. See also Agar plates.

10% SDS

Working solution	Component	Amount per liter
10% (w/v) SDS	SDS	100 g

Denaturing solution

Working solution	Component	Amount per liter
0.5 M NaOH	NaOH	20 g
1.5 M NaCl	NaCl	87.7 g

Neutralization solution

Working solution	Component	Amount per liter
1.5 M NaCl	NaCl	87.7
0.5 M Tris base	Tris base	60.6 g

20x SSC

Working solution	Component	Amount per liter
3 M NaCl	NaCl	175.3 g
0.3 M sodium citrate	Sodium citrate·2H₂O	88.2 g

Agar plates

Prepare LB medium according to the composition given in the table “LB media”. Just before autoclaving, add 15 g agar per liter and mix. After autoclaving, swirl the medium gently to distribute the melted agar evenly throughout the solution. Take care that the hot liquid does not boil over when swirled.

LB media

Component	Amount per liter
Tryptone	10 g
Yeast extract	5 g
NaCl	10 g

Tip: Cool autoclaved agar medium to below 50°C (when you can hold it comfortably) before adding heat-sensitive antibiotics and nutrients. Mix thoroughly to obtain an even concentration throughout the medium before pouring.

Tip: Pour plates in a laminar-flow hood or, if no hood is available, on a cleaned bench surface next to a Bunsen. Use 30–35 ml medium per standard 90 mm petri dish (~30 plates per liter of medium).

After pouring plates, any air bubbles may be removed by passing the flame of a Bunsen burner briefly over the surface. Do not linger with the flame as this may destroy antibiotics in sections of the plates.

Dry plates either directly after solidification or just before use by removing the lids and standing the plates in a laminar-flow hood for 1 hour. Alternatively, if you do not have access to a hood, plates can be dried with the covers slightly open in a 37°C incubator for 30 min, or left upside down with lids on at room temperature for 2–3 days.

Tip: Store plates inverted at 4°C in a dark room or wrapped in aluminum foil to preserve light-sensitive antibiotics. Do not store for longer than 3 months as antibiotics may degrade

Procedure

Plate freshly transformed cells on LB-agar plates containing the appropriate antibiotics, and incubate overnight.

Tip: After spreading the transformation mix, dry the plates inverted with the lids slightly open until small wrinkles develop on the surface of the agar. To prevent smearing, incubation should not be started until all of the liquid has been absorbed into the agar.

Tip: To avoid expression of toxic proteins in the absence of an inducer (a result of “leaky” promoters) and to maintain plasmid stability, incubation can be carried out at 30°C.

Tip: If the expressed protein is not toxic and the plasmids are stable, incubation can be carried out at 37°C, but care should be taken that the colonies do not become too large.
Remove the plates from the incubator, open lids slightly, and allow any condensation to dry for 10 min.
Place a dry, numbered nitrocellulose filter on the agar surface in contact with the colonies, taking care not to introduce air bubbles.

Tip: Hold the filter on opposite sides with blunt-ended forceps, and lower gently onto the agar surface, making contact first along the middle and then lowering (but not dropping) the sides.

Tip: Number filters with a water-resistant marking pen or pencil.
Using a syringe needle, pierce the filter and agar at asymmetric positions to facilitate proper alignment following detection. Grip filter on the sides with blunt-ended forceps, and peel it off in one movement.
Transfer filter (colony side up) to a fresh LB-agar plate and induce expression, e.g., by using a plate containing antibiotics and 250 µM IPTG (see “Induction of protein expression”). Avoid introducing air bubbles.

Tip: Hold the filter on opposite sides with blunt-ended forceps, and lower gently onto the agar surface, making contact first along the middle and then lowering (but not dropping) the sides.
Incubate plates for 4 h at 37°C. Place master plates in a 30°C incubator for 4 h to allow colonies to regrow
Prepare a set of polystyrene dishes for colony lysis and binding of protein to the filters. Each dish should contain a sheet of Whatman 3MM paper soaked with the following solutions:

Dish 1. 10% SDS solution
Dish 2. Denaturing solution
Dish 3. Neutralization solution
Dish 4. Neutralization solution
Dish 5. 2x SSC

Note: Discard excess fluid so that paper is moist but not wet. Excess liquid promotes colony swelling and diffusion and will result in blurred signals.
Place the nitrocellulose filters (colony side up) on top of the paper in each of these dishes, taking care to exclude air bubbles (colonies above air bubbles will not lyse properly and will generate a higher background in the final staining step). Incubate sequentially in the dishes (prepared in step 7), at room temperature as follows:

Dish 1. 10% SDS solution 10 min
Dish 2. Denaturing solution 5 min
Dish 3. Neutralization solution 5 min
Dish 4. Neutralization solution 5 min
Dish 5. 2x SSC 15 min
Continue with the protocol for immunodetection using a chromogenic substrate (see “Immunodetection using a chromogenic method”).

Tip: Due to the problem of high background, protocols using chemiluminescent substrates are not recommended for detection after colony blotting.

Note: At times there is only a slight difference between colonies which express protein and those that do not.

Tip: Shorter staining times are required with this procedure. A 2–3 min staining time is usually sufficient, but it is very important to monitor color development at this stage.

Tip: If it is still difficult to differentiate between positive clones and background, the cause of the high background should be determined.

The following controls should be included:

A plate of host bacteria without the expression plasmid
A plate of host bacteria harboring the expression plasmid without the insert
A colony-blot treated only with secondary antibody prior to detection
A positive control expressing the protein of interest, if possible