Broeck, H.C., van den

English

Wageningen Yield 

Visualising Wheat Gluten Proteins
Staining Profile Depends on the Method Used
Bread Wheat
Wheat is a major food crop used for the 
preparation of bread and pasta. Wheat 
gluten extracts are increasingly used as 
an additive in many food products such 
as soups, sauces, candies, ice cream etc. 
Nowadays, wheat is bred for high qual-
ity, high yield, and disease resistance, 
which has led to thousands of different 
wheat varieties. High quality of bread 
wheat is determined by composition of 
gluten proteins. These proteins have the 
capability to form a matrix by inter- and 
intra-molecular disulfide linkages. Dur-
ing dough preparation, air is captured 
within this matrix, which gives bread its 
volume and structure [1].
Gluten Proteins
Gluten proteins from wheat are com-
posed of monomeric gliadins and poly-
meric glutenins. Gluten proteins are 
composed of a large number of the amino 
acids proline and glutamine and only a 
small number of arginine, lysine, and 
histidine [2]. Gliadins have the charac-
teristic to be soluble in alcohol solutions 
and glutenins are soluble in alcohol solu-
tions containing a reducing agent and in 
diluted acid or alkali [3]. A negative as-
pect of gluten proteins from wheat, rye, 
Hetty C. van den Broeck, 
Senior Assistant Scientist, 
Plant Research International
Gluten proteins from wheat are important for the determination of dough qual-
ity concerning bread making. A negative aspect of these proteins is that they 
can contain peptide sequences that can cause coeliac disease. These gluten pro-
teins are composed of high amounts of the amino acids proline and glutamine, 
which can influence the staining behaviour. A combination of staining methods 
is necessary to identify proteins involved in celiac disease.
and barley is that they can 
contain immune responsive 
peptide sequences that can 
cause coeliac disease (CD). 
After consumption of gluten-
containing foods, these pep-
tides trigger an immune re-
sponse that causes damage to 
the small intestine in CD-pa-
tients. This leads to a range of 
symptoms such as diarrhoea 
and malabsorption [4]. 
Protein Staining Methods
Protein staining methods dif-
fer in the interaction of the 
dye with the amino acids of 
the protein, the detection sen-
sitivity, whether the method is 
end-staining, how the stain is 
detected and in the complex-
ity of the method. Most meth-
ods are based on staining pro-
teins after separation by gel 
electrophoresis, either by so-
dium dodecyl sulphate poly-
acrylamide gel electrophore-
sis (SDS-PAGE) or by 
2-dimensional gel electropho-
resis (2-DE). Traditional Coo-
massie Brilliant Blue (CBB) 
R-250 staining is a simple 
method and interacts with 
lysine and arginine residues 
[5]. The procedure has a low 
detection sensitivity but is 
compatible with mass spec-
trometry. Colloidal staining 
with CBB G-250 (PageBlue) is 
much more sensitive than tra-
ditional CBB R-250 staining 
and does not require a 
destaining step. It is an end-
point staining and interacts 
with lysine, arginine, histi-
dine, and tyrosine residues 
[5]. The silver staining proce-
dure is quite complex and 
over-development can occur. 
It interacts with lysine resi-
dues [5] and provides high 
sensitivity. Sypro Ruby is a 
fluorescence-based end-point 
stain with a high sensitivity 
and is compatible with mass 
spectrometry. The method is 
easy and interacts with lysine, 
arginine, and histidine [5]. An 
image is obtained by using a 
fluorescence scanner. CyDye 
DIGE fluor minimal dyes (Cy2, 
Cy3, and Cy5) are used in Dif-
ference in Gel Electrophore-
sis (DIGE) for labelling differ-
ent protein samples prior to 
analysing them on the same 
gel [6]. This makes it possible 
to detect and quantify differ-
ences between experimental 
pairs of samples resolved on 
the same gel, and between 
gels. The method labels lysine 
residues, is very sensitive [7], 
and images are obtained by 
scanning the different fluo-
rescence dyes. Immunoblot-
ting is also a very sensitive 
detection method. Proteins 
are transferred to a mem-
brane and specific proteins 
can be stained by using anti-
bodies raised against these 
proteins.
Staining Efficiency of Wheat 
Gluten Proteins
Gluten proteins were ex-
tracted from different wheat 
varieties and separated by gel 
electrophoresis. For details of 
the used methods, see [8]. Be-
cause of similar molecular 
masses, many gliadins share 
a mobility region with low 
molecular weight glutenin 
subunits (LMW-GS) if analy-
sed by SDS-PAGE (fig. 1). 
2-DE results in increased res-
olution of these proteins (fig. 
2). All CBB R-250 stained glu-
ten proteins destained to 
some extent, as was already 
shown before [9]. The dura-
tion of destaining showed to 
be important for the resulting 
pattern (fig. 1A and 1B, boxed 
protein bands). When using 
CBB G-250, some proteins 
were poorly stained (fig. 1C, 
marked with arrows). Using 
silver nitrate staining, high 
molecular weight glutenin 
subunits (HMW-GS) were 
poorly stained compared to 
other gluten proteins (fig. 1D). 
The duration of development 
had to be increased, leading 
to over-development. Sypro 
Ruby resulted in better stain-
ing of HMW-GS (fig. 1E), how-
ever, with a speckled back-
ground staining. 
As can be expected, immu-
noblotting using specific anti-
bodies against CD immune 
responsive peptides showed 
that similarly abundant pro-
teins of the same molecular 
weight can react different in 
immune staining, and that 
search the most important criterion in 
the selection of the staining method is 
that as many gluten proteins as possible 
should be visualised, including proteins 
present in small amounts, as all of them 
may contain CD immune responsive 
epitopes.
Acknowledgements
This review is based on collaboration 
with Twan America, René Smulders, 
Luud Gilissen and Ingrid van der Meer 
[8]. IvdM is thanked for critical reading 
the manuscript and editorial comments. 
The research was supported by the  Celiac 
Disease Consortium; an Innovative Clus-
ter approved by the Netherlands Genom-
ics Initiative, partially funded by the 
Dutch Government (BSIK03009), and 
was performed in collaboration with 
Plant Proteomics Hotel facility of Nether-
lands Proteomics Centre. Many thanks to 
Prof. Frits Koning and Dr. Liesbeth 
Dekking for providing us with the mAb 
against T-cell epitope Glia-α20. 
References
[1] Payne P.I.: Ann. Rev. Plant Physio. 38, 
141–153 (1987)
[2] Gianibelli M.C. et al.: Cereal Chem. 78, 
636–646 (2001)
[3] Osborne T.B.: Carnegie Institution of Wash-
ington, Publication 84, 1907
[4] Sollid L.M.: Nat. Rev. Immunol. 2, 647–655 
(2002)
[5] Steinberg T. et al.: Electrophoresis 21, 
486–496 (2000)
[6] Ünlü M. et al.: Electrophoresis 18, 2071–2077 
(1997)
[7] Tonge R. et al.: Proteomics 1, 377–396 
(2001)
[8] Van den Broeck H.C. et al.: Proteomics 8, 
1880–1884 (2008)
[9] Hurkman W.J. and Tanaka C.: J.Cereal Sci. 
40, 295–299 (2004)
ContACt:
Hetty C. van den Broeck
Plant Research International
Wageningen, The Netherlands
hetty.busink@wur.nl
www.pri.wur.nl
Tel.: +31 317 480974
Fax: +31 317 418094
Fig. 1: SDS-PAGE (10 %) analyses of gluten protein extracts from hexaploid wheat varieties. 1. Bovictus; 
2. Combi; 3. Sperber; 4. Rektor; 5. toronto; 6. Ambras. Stained with: A) 15 µg protein, CBB R-250 and 2 
hours destaining. B) 15 µg protein, CBB R-250 and 16 hours destaining. C) 2 µg protein, CBB G-250 
(PageBlue). Arrows indicate poorly stained proteins. D) 1 µg protein, silver nitrate. E) 1 µg protein, 
Sypro Ruby. F) Immunoblot using monoclonal antibody against t-cell epitope Glia-α20.
Fig. 2: 2-DE of gluten proteins from hexaploid wheat variety Rektor, 50 µg protein labelled with Cy3 
combined with 100 µg unlabeled protein. IEF performed on 24 cm linear IPG strip pH 3-10, followed 
by SDS-PAGE (10 %). A) CBB R-250. B) Sypro Ruby. C) Cy3 signal. Red arrowheads represent spots 
selectively visualised with CBB R-250 and Sypro Ruby. Green circles represent spots detected with Cy3. 
the spot indicated with the blue square is detected with Sypro Ruby and Cy3, but not with CBB R-250. 
D, E, and F) Enlargements of boxes in A, B, and C, respectively.
Conclusion
For visualising gluten proteins, all stain-
ing methods described here are applica-
ble, though prominent selective staining 
is observed for each method. Because of 
the aberrant amino acid composition 
and solubility of wheat gluten proteins, 
the staining method used to visualise 
the proteins in 1D or 2D gels affects the 
resulting protein pattern. In CD re-
some low abundant proteins have a 
strong immune response (fig. 1F). 
Because gluten proteins contain only 
small amounts of lysine, arginine, and 
histidine, the efficiency of CyDye label-
ling of specific protein spots showed to 
be affected (fig. 2). Some gluten proteins 
were not labelled (fig. 2) and, on the 
other hand, some other proteins were 
 labelled that were not visualised by  Sypro 
Ruby or CBB R-250. 
BIOforum Europe 9/2008, pp 18–20, GIT VERLAG GmbH & Co. KG, Darmstadt, Germany www.gitverlag.com    www.pRO-4-pRO.com


Visualising wheat gluten proteins : staining profile depends on the method used

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