J. Biochem. Biophys. Methods 64 (2005) 76–81

www.elsevier.com/locate/jbbm

Short note

Detection of neuronal growth inhibitory factor

(metallothionein-3) in polyacrylamide gels and

by Western blot analysis

Gabriele Meloni, Markus Knipp, Milan Vasak*

Institute of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland

Received 23 December 2004; received in revised form 6 May 2005; accepted 27 May 2005

Abstract

Neuronal growth inhibitory factor (GIF) is a small cysteine-rich metal binding protein

downregulated in Alzheimer’s disease. The protein belongs to the superfamily of metallothioneins

(MTs) and was classified as MT-3. Although first identified as a brain specific protein, several

reports now indicate a substantially broader expression pattern. However, currently available

detection methods for MT-3 show low sensitivity in gel electrophoresis and Western blot. We have

developed a fast and sensitive method for MT-3 detection in SDS–PAGE (detection limit ~10 ng)

and Western blot (detection limit ~1 ng). The method is based on the chemical modification of

cysteine residues with the dye monobromobimane and an improved blotting protocol.

D 2005 Published by Elsevier B.V.

Keywords: Metallothionein-3; Growth inhibitory factor; SDS–PAGE; Western blot; Thiol-labeling;

Monobromobimane

0165-022X/$

doi:10.1016/j.j

Abbreviati

phosphate p-to

acid; IPTG, is

chloride; TCE

* Correspond

E-mail add

- see front matter D 2005 Published by Elsevier B.V.

bbm.2005.05.005

ons: AD, Alzheimer’s disease; AP, alkaline phosphatase; BCIP, 5-bromo-4-chloro-3-indolyl

luidine salt; DTT, 1,4-dithio-dl-threitol; Hepes, 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic

opropyl h-d-1-thiogalactopyranoside; mBrB, monobromobimane; NBT, p-nitro-blue tetrazolium

P, tris(2-carboxyethyl)phosphine hydrochloride.

ing author. Tel.: +41 44 6355552; fax: +41 44 6355905.

ress: mvasak@bioc.unizh.ch (M. Vasak).

G. Meloni et al. / J. Biochem. Biophys. Methods 64 (2005) 76–81 77

Neuronal growth inhibitory factor (GIF) is a small (~7 kDa) cysteine-rich metal binding

protein downregulated in Alzheimer’s disease (AD) [1]. It belongs to the superfamily of

metallothioneins (MTs) and was classified as metallothionein-3 (MT-3) (reviewed in Ref.

[2]). The protein was discovered based on its ability to antagonize the neurotrophic effect

of AD brain extracts to stimulate survival and neuritic sprouting of cultured neurons. MT-3

consists of 68 amino acids, 20 of which are Cys, and binds metal ions such as Zn2+ and

Cu+ with a high affinity into two protein domains. Although MT-3 has been first identified

as the brain specific isoform, its expression has since then been reported in other organs

and cancer cells [3]. Therefore, there is an increasing interest in the detection and

quantification of MT-3 in tissues and cell lines. Because of inherent difficulties, which

accompany its detection at the protein level, MT-3 was usually monitored only through its

mRNA.

Besides a general low detection sensitivity of all MTs with common laboratory methods

like SDS–PAGE and Western blot, the detection of MT-3 is associated with a band

broadening in SDS–PAGE, reduced electrophoretic mobility, and low binding to blotting

membranes. This behavior presumably reflects its high hydrophilicity and negative charge

(charge of the apoprotein: �12 at pH 8.3), characteristics that are only partially shared by

other members of the MT superfamily. Although a number of various methods for MT

detection by Western blot analysis have been described (Ref. [4] and references therein),

the reported sensitivity for MT-3 was always substantially lower compared to the other MT

isoforms. This unusual behavior often requires sample concentration prior to Western blot

analysis making MT-3 detection in biological samples difficult and time consuming.

Here we describe a fast and sensitive method for the detection of MT-3 in SDS–PAGE

and by Western blot analysis. The method is based on Cys modification with the

fluorescence dye monobromobimane (mBrB) [5]. This modification of MT-3 gave rise to

sharp bands in gel electrophoresis and an improved blotting efficiency. Modification with

mBrB has proven useful in specific labeling of both proteins and low molecular weight

thiols. The compound itself is only weakly fluorescent but upon reaction with a thiol, it

forms a highly fluorescent and stable thioether that can be easily detected at the picomolar

level. Due to the high Cys content of MT-3 and the hydrophobicity of the introduced

mBrB molecules, the properties of the labeled protein are significantly altered. This results

in a highly improved detection of MT-3 in SDS–PAGE and Western blot.

The procedure was developed using recombinant human Zn-MT-3 expressed in

Escherichia coli and purified as described [6]. Fully Zn2+ loaded MT-3 (Zn7MT-3) was

prepared according to Refs. [6,7]. The free Cys content was controlled by the sulfhydryl

reaction with 2,2V-dithiodipyridine [6]. Stock solutions of Zn7MT-3 (20 or 5 ng/Al) wereprepared in 10 mM Hepes/NaOH, 100 mM KCl, 1 mM DTT, 0.5 mM MgCl2, pH 7.4. The

mBrB modification of Cys was performed with Zn7MT-3 samples (0.1–10 Al of stocksolution) to which 10 Al of 60 mM Tris/HCl, 20 mM EDTA, 20 mM TCEP, pH 8.0 were

added. Subsequently, the solution was mixed with 2.5 Al of 100 mM mBrB (Fluka GmbH,

Switzerland) in CH3CN. Samples were incubated at room temperature for 1 h in the dark1.

Afterwards, excess mBrB was removed by extraction with 150 Al of CH2Cl2. Samples

1 Free bromobimanes are sensitive to light yielding a fluorescent product.

G. Meloni et al. / J. Biochem. Biophys. Methods 64 (2005) 76–8178

were briefly mixed 3 times on a Vortex and immediately spun down. CH2Cl2 (lower

phase) was carefully removed and the extraction step repeated twice. It should be noted

that the extraction affects the detection limit only marginally but it improves the visual

quality of SDS–PAGE.

Prior to SDS–PAGE 5 Al of 10 mM Tris/HCl, 10 mM EDTA, 20% (v/v) glycerol, 1%

(w/v) SDS, 0.025% (w/v) bromophenol blue, 100 mM DTT, 0.7 M 2-mercaptoethanol, pH

7.5 were added to the modified protein and the samples boiled for 5 min at 95 8C. Thesamples were run on 15% mini-SDS–PAGE (8.3�7.3 cm, prepared according to Ref. [8])

at constant voltage (200 V) on a MiniProtean apparatus (BioRad). Gels were stained either

with 1% (w/v) Coomassie Brilliant Blue R-250 in 40% CH3OH/10% CH3COOH/50%

H2O or washed 5 min in H2O prior to fluorescence imaging. To further improve the quality

of fluorescence image, unspecific fluorescence caused by excess mBrB was removed by

an additional incubation in 40% CH3OH/10% CH3COOH/50% H2O for 1 h. For

detection a fluorescence imager (GeneGenius, Syngene, Cambridge, UK) equipped with a

UV transilluminator (302 nm) and an emission filter (500–600 nm) placed before the

camera objective was used.

For Western blot analysis a slightly modified procedure of Mizzen et al. [4] was

adopted. Gels and 0.2-Am pore nitrocellulose membranes (Schleicher and Schuell, Dassel,

Germany) were incubated in transfer buffer (25 mM Tris/HCl, 192 mM glycine, 2 mM

CaCl2, 20% CH3OH, pH 8.3) at 4 8C for 15 min. Blotting was performed at constant

voltage (40 V) at 4 8C for 2.5 h. Afterwards, the membrane was treated as described in

Ref. [4] and incubated with the anti-hMT-3a raised against an a-domain specific peptide

of MT-3 in rabbits [9]. An alkaline phosphatase (AP) linked goat-anti-rabbit IgG (Sigma-

Aldrich Co.) was used and the blot developed using BCIP and NBT as AP substrates.

The applicability of the method has also been tested on complex protein samples. E.

coli (strain: BL21 (DE3) pLysSCam, Novagen) extracts were obtained from 1.5 ml of

isopropyl h-d-1-thiogalactopyranoside (IPTG) induced and non-induced cultures contain-

ing the human pEThMT3Amp expression vector [6]. Bacterial pellets were resuspended in

100 Al of 50 mM Tris/HCl, 100 mM NaCl, 60 mM 2-mercaptoethanol, pH 8.0 and

sonicated. Cell debris was removed by centrifugation (15,000 �g, 20 min, 4 8C). Thesoluble protein fraction of human frontal brain lobe was prepared through homogenization

with 2 :1 (w/v) 50 mM Tris/HCl, pH 7.6, in a Potter homogenizer. Cell debris were

removed through centrifugation (100,000 �g, 1 h, 4 8C) and protease inhibitors were

added (Complete Protease Inhibitor Cocktail, Roche). Both homogenates were treated

according to pure protein samples except that 10 Al of 100 mM mBrB in CH3CN was

added. After the CH2Cl2 extraction the precipitated protein was removed by centrifugation

(15,000 �g, 2.5 min, room temperature).

In the method reported here, the modification of MT-3 led to a sharp band in SDS–

PAGE, increased blotting efficiency, and higher immunoblotting sensitivity. Previously,

gradient gels were used to decrease the band width of MT-3 [10]. In our studies, we

employed the recently reported fast and easy preparation of polyacrylamide gels without

stacking gel [8], which extremely simplified the protein electrophoresis. In Fig. 1A the

mobility profiles of MT-3 and mBrB-MT-3, both stained with Coomassie Brilliant Blue,

are shown. In the absence of Cys modification MT-3 runs as a broad and diffuse band with

an apparent molecular mass of ~22 kDa (apoMT-3: ~7 kDa). The limit of protein detection

Fig. 1. (A) SDS–PAGE of purified MT-3 (from 100 ng to 1000 ng) without (upper panel) and upon mBrB

modification (lower panel) stained with Coomassie Brilliant Blue R-250 (marker proteins: carbonic anhydrase, 31

kDa; trypsin inhibitor, 21.5 kDa; lysozyme, 14.4 kDa). (B) MT-3 can be visualized directly in gels (without any

staining procedure) by fluorescence imaging. (C) Fluorescence detection of recombinant mouse MT-1 (prepared

as described [12]) modified with mBrB (lane 1, 100 ng), mBrB-MT-3 (lane 2, 100 ng), and a mixture of mBrB-

MT-1 and mBrB-MT-3 (lane 3, 100 ng each).

G. Meloni et al. / J. Biochem. Biophys. Methods 64 (2005) 76–81 79

lies in the range of ~500 ng. In marked contrast, mBrB-MT-3 shows a rather sharp band

with an apparent molecular mass of ~10 kDa, reflecting the mass increase upon

modification. In addition, a greatly improved detection limit of ~100 ng was obtained.

Taking advantage of the introduced fluorescence groups the modified protein can also be

visualized directly in the gel by fluorescence imaging [11]. The fluorescence analysis of

MT-3 shifted the detection limit down to ~10 ng (Fig. 1B). The method is also applicable

to other metallothionein isoforms, i.e., MT-1 and MT-2. In addition, it allows the

separation of these isoforms from about 15% larger MT-3. This is exemplified by gel

electrophoresis of a mixture of MT-1 and MT-3 (Fig. 1C).

For immunodetection of MT-3 and analysis in crude biological samples the Cys

modification protocol was combined with an improved blotting procedure for MT [4]. The

G. Meloni et al. / J. Biochem. Biophys. Methods 64 (2005) 76–8180

alteration of MT-3 properties upon modification resulted in its enhanced retention on

nitrocellulose membrane. Thus, compared to previous reports [4] mBrB-MT-3 can be

blotted with a much higher efficiency. This improved both quality and sensitivity of the

immunodetection (Fig. 2). As shown in Fig. 2A the detection limit in Western blot analysis

is ~1 ng when an AP-conjugate secondary antibody was used. In addition, the

modification of Cys allows easy monitoring of the blotting efficiency by a direct

Fig. 2. (A) Western blot analysis of mBrB modified MT-3 (lower panel) compared to unmodified protein (upper

panel), using an AP-conjugate detection system. Equal amounts of protein between 200 ng and 1 ng were loaded

on both gels. (B) Western blot of MT-3 upon mBrB modification in biological samples: lane 1 and 5, 50 ng of

purified MT-3 as a control; lane 2, 5 Al of E. coli soluble protein extract, IPTG induced; lane 3, 5 Al of E. colisoluble protein extract, not induced; lane 4, 5 Al of soluble extract of human frontal brain lobe. (Pre-stained

marker proteins (National Diagnostic): ovalbumin, 47 kDa; carbonic anhydrase, 25 kDa; trypsin inhibitor, 22

kDa; lysozyme, 17 kDa).

G. Meloni et al. / J. Biochem. Biophys. Methods 64 (2005) 76–81 81

fluorescence imaging of the membrane prior to Western blot analysis. This is feasible for

MT-3 down to the low nanogram range (data not shown).

In order to demonstrate the applicability of this method to tissue samples and cell

extracts Western blot analysis of MT-3 in homogenate of human brain and E. coli extract

were performed (Fig. 2B). In these instances, no further protein fractionation or purification

step was required. Moreover, serial dilutions of brain homogenate analyzed by Western blot

resulted in a serial signal decrease as observed for the pure protein. The addition of isolated

MT-3 to brain homogenate resulted in its complete recovery (data not shown).

In conclusion, the method described is highly suitable for the detection and

quantification of MT-3 in various biological samples. Moreover, it may prove useful in

increasing immunoblotting detection of other MTs and small hydrophilic proteins that

contain a significant amount of Cys residues.

Acknowledgements

Authors thank Dr. G. Fritz (University of Konstanz, Germany) for kindly providing

human brain extract samples. This work was supported by the bForschungskommission

und Nachwuchsforderungskommission der Universitat ZurichQ (to M. K.), by Swiss

National Science Foundation Grant 3100A0-100246/1 (to M.V.) and the bJubilaumsspende

der Universitat ZurichQ (to M.V.).

References

[1] Yu WH, Lukiw WJ, Bergeron C, Niznik HB, Fraser PE. Metallothionein III is reduced in Alzheimer’s

disease. Brain Res 2001;894:37–45.

[2] Hidalgo J, Aschner M, Zatta P, Vasak M. Roles of the metallothionein family of proteins in the central

nervous system. Brain Res Bull 2001;55:133–45.

[3] Sens MA, Somji S, Garrett SH, Beall CL, Sens DA. Metallothionein isoform 3 overexpression is associated

with breast cancers having a poor prognosis. Am J Pathol 2001;159:21–6.

[4] Mizzen CA, Cartel NJ, Yu WH, Fraser PE, McLachlan DR. Sensitive detection of metallothioneins-1, -2 and

-3 in tissue homogenates by immunoblotting: a method for enhanced membrane transfer and retention. J

Biochem Biophys Methods 1996;32:77–83.

[5] Kosower NS, Kosower EM. Thiol labeling with bromobimanes. Methods Enzymol 1987;143:76–84.

[6] Roschitzki B, Vasak M. Redox labile site in a Zn4 cluster of Cu4,Zn4-metallothionein-3. Biochemistry

2003;42:9822–8.

[7] Vasak M. Metal removal and substitution in vertebrate and invertebrate metallothioneins. Methods Enzymol

1991;205:452–8.

[8] Ahn T, Yim SK, Choi HI, Yun CH. Polyacrylamide gel electrophoresis without a stacking gel: use of amino

acids as electrolytes. Anal Biochem 2001;291:300–3.

[9] Roschitzki B, Vasak M. A distinct Cu4-thiolate cluster of human metallothionein-3 is located in the N-

terminal domain. J Biol Inorg Chem 2002;7:611–6.

[10] Uchida Y, Takio K, Titani K, Ihara Y, Tomonaga M. The growth inhibitory factor that is deficient in the

Alzheimer’s disease brain is a 68-amino acid metallothionein-like protein. Neuron 1991;7:337–47.

[11] Fan TWM, Lane AN, Higashi RM. An electrophoretic profiling method for thiol-rich phytochelatins and

metallothioneins. Phytochem Anal 2004;15:175–83.

[12] Romero-Isart N, Jensen LT, Zerbe O, Winge DR, Vasak M. Engineering of metallothionein-3

neuroinhibitory activity into the inactive isoform metallothionein-1. J Biol Chem 2002;277:37023–8.