36
L. CASTELLANOS-SERRA et al.
2.5
In-Gel Renaturation
Procedure (Hardy et al. 1996)
Stain the gel as described under 2.2.1, excise the bands of interest and incubate in
Tris-EDTA buffer (see 2.3) to chelate zinc 2+.
In-gel renaturation: Wash the gel slices twice in PBS (1 mL) for 10 min to remove
excess EDTA. Soak the gel slice (3 xI mL x 10 mins) in PBS containing 0.1 % Triton X-IOO, and next, wash twice in PBS alone (1 mL), to remove excess detergent.
Protein elution: Proceed as described under 2.6.
Comments
i) The present approach shares some similarities with protein renaturation
accomplished in gel-filtration matrices, through buffer exchange from denaturing to non-denaturing conditions. As in such cases, there is not a unique
protocol for protein renaturation, therefore, it may be necessary to explore a
number of renaturing conditions. This protocol can serve as a guide for further optimization, as it has proved useful for a number of proteins both for
determination of specific activity after recovery of the renatured protein in
solution and for zymogram detection on gel. For optimization, the following
factors can be modified: detergent exchanged with SDS (NP-40, sodium
deoxycholate, Triton X-IOO, octyl glucoside), incubation time (from minutes
to h) and temperature (4°C, 20 °C, 37°C) during the renaturing step. Alternatively, in cases where the protein is sensitive to detergents, the gel slice can be
incubated in 30 % acetonitrile in PBS (2 x 10 mins) to remove SDS, followed
by incubation in PBS.
ii) Protein renaturation can be accomplished in the entire gel, which can then be
analyzed by zymogram detection.
iii) Proteins should not be reduced before electrophoresis, to facilitate the recovery of biological activity.
iv) For determining specific activity, several bands may be processed in order to
get enough material for both protein quantitation and biological assay. Each
determination should be done at least in triplicate.
v) Whenever a biological assay is attempted, carry out a control experiment in
parallel, by processing an equal number of protein-free gel bands. This permits to evaluate any potential toxicity or interference derived from gel and
electrophoresis reagents.
vi) This procedure obviously does not work for proteins formed by several subunits of different molecular weight which are separated on the gel, if the biological activity requires the assembly of a heteromeric structure. In this case,
protein elution (see 2.6) can be directly performed after separation and
detection under native conditions.
L. CASTELLANOS-SERRA et al.
2.5
In-Gel Renaturation
Procedure (Hardy et al. 1996)
Stain the gel as described under 2.2.1, excise the bands of interest and incubate in
Tris-EDTA buffer (see 2.3) to chelate zinc 2+.
In-gel renaturation: Wash the gel slices twice in PBS (1 mL) for 10 min to remove
excess EDTA. Soak the gel slice (3 xI mL x 10 mins) in PBS containing 0.1 % Triton X-IOO, and next, wash twice in PBS alone (1 mL), to remove excess detergent.
Protein elution: Proceed as described under 2.6.
Comments
i) The present approach shares some similarities with protein renaturation
accomplished in gel-filtration matrices, through buffer exchange from denaturing to non-denaturing conditions. As in such cases, there is not a unique
protocol for protein renaturation, therefore, it may be necessary to explore a
number of renaturing conditions. This protocol can serve as a guide for further optimization, as it has proved useful for a number of proteins both for
determination of specific activity after recovery of the renatured protein in
solution and for zymogram detection on gel. For optimization, the following
factors can be modified: detergent exchanged with SDS (NP-40, sodium
deoxycholate, Triton X-IOO, octyl glucoside), incubation time (from minutes
to h) and temperature (4°C, 20 °C, 37°C) during the renaturing step. Alternatively, in cases where the protein is sensitive to detergents, the gel slice can be
incubated in 30 % acetonitrile in PBS (2 x 10 mins) to remove SDS, followed
by incubation in PBS.
ii) Protein renaturation can be accomplished in the entire gel, which can then be
analyzed by zymogram detection.
iii) Proteins should not be reduced before electrophoresis, to facilitate the recovery of biological activity.
iv) For determining specific activity, several bands may be processed in order to
get enough material for both protein quantitation and biological assay. Each
determination should be done at least in triplicate.
v) Whenever a biological assay is attempted, carry out a control experiment in
parallel, by processing an equal number of protein-free gel bands. This permits to evaluate any potential toxicity or interference derived from gel and
electrophoresis reagents.
vi) This procedure obviously does not work for proteins formed by several subunits of different molecular weight which are separated on the gel, if the biological activity requires the assembly of a heteromeric structure. In this case,
protein elution (see 2.6) can be directly performed after separation and
detection under native conditions.