Centrifuge Blotting Protocol

Overview

Introduction of Centrifuge Blotting Protocol

Centrifuge immunoblotting is a technique developed by Hermansen et al. to overcome the disadvantages with the downstream steps associated with sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE). This technique alleviates problem, such as low protein recovery, presence of contaminating substances from polyacrylamide gel, fixation and denaturation or blockage of N terminus due to the staining and destaining steps involved, to name a few.

Briefly, the protein samples are prepared and separated by SDS-PAGE as described. Following electrophoresis the gel is soaked in 1 M KCl for 2 min to visualize the protein bands (clear zone against an opaque background). The gel pieces were then used for centrifuge blotting onto the PVDF membranes. PVDF membranes with immobilized proteins could be used for N-terminal sequence analysis or vacuum-dried and stored at −20 °C until further analysis.

Some advantages of centrifuge blotting over the existing techniques are the following: protein concentrations in subnanomolar range can easily be eluted within 1–2 h for Edman degradation. Lack of staining and destaining steps prevents N-terminal blockage, the small size of the PVDF membrane makes it suitable for direct application for microsequencer, and positioning of the dialysis membrane directly beneath the PVDF membrane retains the non-immobilized proteins.

Materials of Centrifuge Blotting Protocol

Detection of Protein Bands in the Gel

1 M KCl.
Distilled water.

Centrifuge Blotting

PVDF membrane (12 mm diameter) (see Note 8).
3–10 kDa dialysis membrane discs cut into 12 mm diameter (depending on the size of the protein).
100 % methanol.
Eluant solution (0.05 % SDS/5 % methanol/0.05 % DTT) (see Note 1).

Centrifuge Receptacle Assembly

Outer cylinder constructed of polyoxymethylene (supports whole structure).
Inner cylinder constructed of tetrafluoroethylene (reservoir for the eluent).
12 mm polyethene sinter base support (holds dialysis and PVDF membranes in place).
O-ring silicone gasket (prevents leakage in receptacle).
Polystyrene tube (collection of eluate).

Centrifugation

Hettich EBA 3S tabletop centrifuge or Sorvall RC5C automatic superspeed refrigerated centrifuge with fixed-angle SS-34 rotor.

Methods of Centrifuge Blotting Protocol

SDS-PAGE

Prepare and separate the protein samples according to the method described.
The dimension of the separation gel is 120 × 120 × 1.5 mm.
The acrylamide N,N′-methylenebisacrylamide concentration of the separation gel is about 12 % T, 3 % C.

Detection of Protein Bands on the Gel

Soak SDS-polyacrylamide slab gel in 1 M KC1 for 2-min bands in gel.
Excise the protein band (clear zone against an opaque background) and soak in distilled water for 3–5 min (can store the moist gel in an Eppendorf tube at −20 °C) to remove excess KCl.

Centrifuge Blotting

Cut the PVDF and dialysis membrane (10 kD molecular weight cutoff) into 12 mm discs.
Soak the PVDF membrane in 100 % methanol for 5 min for activation.
Soak the gel pieces in the eluant solution for 15 min at 37 °C (see Note 2).
Soak the PVDF and dialysis membranes in the eluant solution for 3–5 min prior to centrifugation.
Place the dialysis membrane on the base support of the receptacle followed by the O-ring.
Place the PVDF membrane on top of the O-ring after adding 100 μL eluant to the dialysis membrane. This prevents the vacuum formation between the two membranes.
Screw in the inner cylinder over the receptacle.
Place the gel piece over the PVDF membrane and pipette 300 μL of eluant solution into the receptacle (see Note 3).
Ensure that the assembly is complete.
Centrifuge at 3,000 × g at 20 °C for 1 h (see Notes 4 – 7).

Figure 1. The centrifuge-blotting assembly: The assembly consists of an outer cylinder which supports the whole structure.

After centrifugation is done, remove the PVDF membrane with the adhering protein, vacuum dry, and store at −20 °C until further analyses (see Note 8).

Notes of Centrifuge Blotting Protocol

The use of the eluent solution (0.05 % SDS/5 % methanol/0.05 % DTT) improved the protein yield and secured better adherence of proteins onto the PVDF membranes (methanol weakens the protein-SDS interaction, hence allowing improved adsorption of the protein onto the PVDF membrane).
Equilibrating the gel pieces in the eluent solution improves the overall yield.
It is essential to have direct contact between the gel piece and the PVDF membrane, one gel piece with max size of 10 × 6 × 1.5 mm. The direct contact between the gel piece and the PVDF membrane avoids the endosmosis of protein towards the gel.
Polypeptides less than 30 kDa can be blotted by 1-h centrifugation. Higher molecular weight proteins require longer centrifugation time (>3 h). Greater elution of proteins is achieved by extending the centrifugation time up to 6 h or more.
Centrifuge for longer time at 3,000 × g if dead stop volume is observed.
To avoid dead stop volume, use swinging-bucket rotor instead of fixed-bucket rotor.
Appropriate flow rate is essential for good desalting and efficient blotting. To eliminate the variability in flow rate between different runs, puncture a hole in a dialysis membrane with 0.05 mm thick wire and using two PVDF membrane, the first one right above the dialysis membrane and the second one above the O-ring gasket.
PVDF (Immobilon-P) membrane is resistant to the mechanical strain experienced during centrifugation unlike siliconized glass fiber "glassybond" membrane and Whatman glass fiber impregnated with Polybrene.

Reference