Analytical Biochemistry 611 (2020) 114001

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Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio

Technical note

A proteolytic method for evaluating O-GlcNAcylation on proteins of similar

molecular weight to antibody heavy chain after immunoprecipitation
Miranda Machacek a, b, Patrick E. Fields a, Chad Slawson b, *
a
Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, 66160, USA
b
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS, 66160, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Investigating a protein of interest that runs at the same molecular weight as antibody heavy chain is a frequent
O-GlcNAc deterrent to its evaluation by immunoprecipitation. Methods of minimizing the detection of the immunopreci­
IdeZ protease pitating antibody are available. However, these still present a barrier to evaluating if intracellular proteins are
Immunoprecipitation
modified by the O-GlcNAc post-translation protein modification due to interfering glycosylation on antibodies.
Post-translational protein modification
IdeZ protease specifically cleaves antibody at the hinge region, allowing collapse of the antibody fragments to 25
kDa after denaturation. Thus, this proteolytic method uniquely allows evaluation of O-GlcNAcylation of proteins
of interest formerly obscured by antibody heavy chain.

In immunoprecipitation (IP) reactions, an antibody against the pro­
tein of interest is added to the cell lysate in order to enrich the protein of
interest from the thousands of other proteins in a cell. In this way, the
presence of post-translational protein modifications or protein-protein
interactions may be observed or compared between different treat­
ments. Of course, when the protein of interest is precipitated, the anti­
body is precipitated along with it. After denaturation of the protein-
antibody complex, the antibody heavy and light chains form promi­
nent bands on the Western blot at ~50 and ~25 kDa respectively. Thus,
a common impediment to performing immunoprecipitation is an
inability to see proteins of interest that run in the 40–60 kDa molecular
weight range due to strong signal from the 50 kDa antibody heavy chain
running at a similar molecular weight on Western blot. Initial attempts
aimed at working around these limitations included simply using
different species of antibody for immunoprecipitation and the primary
antibody during the Western blot to reduce cross-reactivity. Others did
not add denaturing reagents in order to maintain the antibody molecular
weight at 150 kDa. However, antibody bands are frequently seen even
with use of different species and lack of denaturation often leads to
incomplete release of the protein of interest, leading to muddied and
inaccurate results. This led to the development of more sophisticated
reagents, including light or heavy chain specific antibodies labeled with
enzymatic (i.e. horseradish peroxidase (HRP)) or fluorescent tags [1],
HRP-conjugated reagent capable of binding non-denatured primary
antibody only [2], or HRP-conjugated Protein A or G beads [3]. These
methods do allow visualization of proteins of interest and
protein-protein interactions by binding differentiating aspects of the
primary antibodies while excluding the denatured heavy and light chain
used for immunoprecipitation. However, the heavy chain remains pre­
sent and its detection is simply minimized. Because of this, none of these
methods also allow for detection of post-translation glycosylation on the
protein of interest, since antibodies are also heavily glycosylated [4].
Thus, determining if a protein of interest is glycosylated by immuno­
precipitation is still problematic if the protein runs at the same molec­
ular weight of antibody bands. Ideally, one could visualize a protein of
interest, any interacting proteins, and determine its glycosylation status
with a single immunoprecipitation method. Here we describe a novel
method of proteolytically digesting immunoprecipitating antibody to
25 kDa fragments in a single, quick step, allowing visualization of pro­
teins of interest—and importantly—their post-translational protein
glycosylation modifications, which were previously unable to be visu­
alized using current immunoprecipitation techniques.
While protein glycosylation is traditionally thought to occur on
secreted proteins, glycosylation of intracellular proteins occurs ubiqui­
tously in the form of O-linked N-acetyl-glucosamine (O-GlcNAc) [5].
O-GlcNAcylation is the addition of a single N-acetyl-glucosamine moiety
on serine and threonine residues of nuclear, cytoplasmic, and mito­
chondrial proteins [6]. Thousands of intracellular proteins are modified
by O-GlcNAc, which can affect protein stability, localization, and ac­
tivity [6]. Of note, the two enzymes regulating the addition and removal

* Corresponding author.
E-mail address: cslawson@kumc.edu (C. Slawson).

https://doi.org/10.1016/j.ab.2020.114001
Received 23 May 2020; Received in revised form 12 October 2020; Accepted 21 October 2020
Available online 28 October 2020
0003-2697/© 2020 Elsevier Inc. All rights reserved.
M. Machacek et al. Analytical Biochemistry 611 (2020) 114001

Fig. 1. IdeZ protease cleaves specifically at the hinge region, allowing for collapse of heavy chain from 50 kDa to 25 kDa and visualization of proteins of
interest that run at a similar molecular weight. A. IdeZ protease cleaves antibody at the hinge region, resulting in generation of F(ab’)2 and Fc fragments. With
disruption of disulfide bridges by denaturing buffer, the bands collapse to 25 kDa. B. Schematic of modified immunoprecipitation procedure with addition of IdeZ
protease incubation step of 30 min. An additional control of primary antibody without IdeZ protease addition provides reassurance that the antibody is fully digested
in samples of interest. The unfilled rectangle represents the protein of interest (POI), and hexagons represent glycosylation present on both the antibody and protein
of interest. The black rectangles represent antibody components (heavy and light chain).

of the modification (O-GlcNAc transferase and O-GlcNAcase respec­
tively) are often found associated with transcription factors, which are
frequently O-GlcNAcylated [7,8]. Unfortunately, many transcription
factors, including nuclear receptor, forkhead, and GATA family tran­
scription factors, are around 50 kDa [9]. This creates a unique challenge
in investigating O-GlcNAcylation of many transcription factors by
immunoprecipitation. Our proteolytic method of cleaving antibody
heavy chain solves the conundrum of evaluating the O-GlcNAcylation
status of proteins of interest that previously have been inscrutable.
Our method utilizes the specificity of IdeZ protease, an IgG endo­
peptidase, isolated from the bacteria Streptococcus equi, subspecies
zooepidermicus [10]. IdeZ protease cleaves specifically at the hinge re­
gion of IgG, creating a F(ab’)2 and Fc fragment [11], which collapses to
protein fragments of 25 kDa when further exposed to denaturing agents
(Fig. 1A). As evidence of the high specificity of the IgG endopeptidases, a
homologous IgG endopeptidase, IdeS, has been successfully transfused
into human patients to cleave antibodies generated against transplanted
organs [12]. Thus, we utilized this high specificity to cleave the
precipitating antibody in our IP samples, leaving cellular proteins intact
and allowing assessment of O-GlcNAcylation of a protein running at a
similar molecular weight to heavy chain (Fig. 1B). Cell lysate prepara­
tion and immunoprecipitation procedures are previously described [13].
Briefly, naïve CD4+ T cells were isolated by flow cytometry from spleens
of 12–20 week-old wild type C57BL/6 mice (Jackson Laboratories). T
cells were then incubated with or without 10 μM Thiamet-G (TMG, a
highly selective inhibitor of the enzyme that removes O-GlcNac [14]) for
six hours and then activated on anti-CD3 and anti-CD28 coated plates in
the presence of cytokines (IL-6 and TGFβ) to drive differentiation to­
wards the T helper 17 (Th17) lineage. After 4 days of culture in IMDM,
Th17 cells were harvested and cell lysate extracted with 20 mM Tris, pH
7.4, 150 mM NaCl, 40 mM GlcNAc, 2 mM EDTA, 1 mM DTT, 1% Nonidet
P-40 lysis buffer with protease inhibitors. One μg of anti-RORγt primary
antibody was added to 500–1000 μg of the cell lysate from Th17 cells
treated with (+) and without (− ) TMG for the IP samples. One μg of
non-specific rabbit IgG antibody was also added to an equivalent
amount of a 1:1 ratio of TMG treated and untreated cell lysate to rule out
non-specific antibody binding. Two extra control samples containing
only lysate buffer spiked with 1 μg of primary antibody to eventually be
treated with and without addition of IdeZ protease were also included.
Treatment of the primary antibody with and without IdeZ protease
serves as assurance that the digestion of the antibody occurs to
completion in the digested samples. Samples were rotated overnight at
4 ◦ C, then rotated with anti-Fc receptor binding beads for two hours at
4 ◦ C, and finally washed vigorously. Per the manufacturer’s instructions
(Promega), the optimal buffer for IdeZ protease activity is 50 mM so­
dium phosphate, pH 6.6, 150 mM NaCl. Thus, after washing IP samples
in high salt buffer sufficient to remove non-specific protein binding, the
washing buffer should be transitioned to a neutral pH buffer with min­
imal salt. In our case, we washed with RIPA buffer containing 1 M NaCl
for three washes and then two washes in PBS. Five units of IdeZ protease
(Promega) were then added to all samples, except the undigested pri­
mary antibody control, and resuspended in PBS to a total volume of 20
μL. After an incubation of 30 min at 37 ◦ C, 5 μL of 5X Laemmli buffer was
added and the samples were boiled for 2 min to fully denature the IP
samples. Samples were then loaded onto SDS-PAGE gels along with 40
μg of cell lysate inputs and developed for Western blot analysis as pre­
viously described [13].
While developing this method, we evaluated a transcription factor of
interest to us, retinoic acid receptor-related orphan receptor gamma t
variant (RORγt). RORγt is the master transcription factor that dictates
the differentiation of CD4+ T cells towards a T helper 17 (Th17) lineage
[15]. Th17 cells are pro-inflammatory CD4+ T cells, which aid in the
eradication of fungal and bacterial infections [16]. However, these in­
flammatory cells are also implicated in autoimmune diseases, such as

2
M. Machacek et al.
multiple sclerosis and rheumatoid arthritis [17,18], as well as the
pathogenesis of chronic inflammatory diseases such type 2 diabetes and
obesity [19,20]. We observed that CD4+ T cells treated with Thiamet-G
(TMG), a highly selective inhibitor of the enzyme that removes the
O-GlcNAc modification (O-GlcNAcase) [14], secreted more of the
pro-inflammatory cytokine, interleukin-17 (IL-17), made by Th17 cells
[13]. We then determined by chromatin immunoprecipitation that TMG
treatment also led to more RORγt remaining bound to the il17 gene
promoter consistent with increased IL-17 secretion. Since O-GlcNAc
regulates many transcription factors, we hypothesized RORγt is
O-GlcNAcylated, and direct O-GlcNAcylation of RORγt is a mechanism
for increased RORγt retention at the il17 gene promoter. Unfortunately,
RORγt’s molecular weight is 57 kDa and thus obscured by heavy chain.
Using our novel proteolytic method, we were successfully able to visu­
alize RORγt (anti-RORγt, Abcam), without interference of the heavy
chain band and determine that it is not O-GlcNAcylated in CD4+ T cells
differentiated towards the Th17 lineage ex vivo (Fig. 2). Thus, we
determined direct O-GlcNAcylation of RORγt is an unlikely mechanism
for its retention at the il17 gene promoter.
Using IdeZ protease to digest heavy chain represents a significant
improvement to current techniques to mask heavy chain bands on
Western blot. It is the only method that allows for evaluation of the
presence of a protein of interest, protein-protein interactions, as well as
modification by O-GlcNAc. In addition to cellular lysates, this technique
could be used on tissue samples with high endogenous antibody binding
being evaluated by Western blot (spleen, lymph nodes, organs in auto­
immune disease models, etc.). Potentially, the presence of the IdeZ
protease could obscure proteins of interest, since a faint band often
appears on the Western blot (Fig. 2). Formulations of IdeZ with a His-tag
(New England Biolabs) are available, which would allow for its removal
by running samples over a nickel column if desired. The high specificity
3
Analytical Biochemistry 611 (2020) 114001
Fig. 2. Heavy chain is completely
cleaved, revealing that RORγt is not O-
GlcNAcylated. A. OGlcNAc levels are
appropriately increased in the input sample
treated with TMG (+) compared to un­
treated sample (− ). However, no O-GlcNAc
signal is detected at 57 kDa where RORγt
runs. Importantly, while heavy chain is seen
in the undigested primary antibody sample
(1◦ ), it is absent in the primary antibody
sample treated with IdeZ protease and there
is a concomitant increase in protein running
in the 25 kDa light chain region due to heavy
chain’s cleavage by the IdeZ protease. A
nonspecific isotype matched antibody (IgG)
was included to rule out non-specific
immunoprecipitation of RORγt. B. RORγt is
successfully and specifically immunoprecip­
itated and present at its predicted molecular
weight of 57 kDa. Heavy chain remains
present in the undigested primary antibody
sample and runs slightly below RORγt but
again is absent in the digested primary
antibody only sample.
of IdeZ protease is ideal for alleviating concerns of off-target digestion of
proteins in the cellular lysate, but also limits the types of antibodies it
can cleave. All human, humanized, rabbit, sheep, monkey, and Fc-fusion
chimeric IgG antibodies can be digested by IdeZ protease. However,
other immunoglobulin subtypes (i.e. IgM, IgA), mouse IgG1, rat,
porcine, bovine, and goat IgG are not able to be cleaved. IdeZ has
reduced activity against mouse IgG2a and IgG3, which may require
longer incubation times to achieve complete digestion. Thus, careful
selection of an appropriate immunoprecipitating antibody is essential
for successful evaluation of a protein of interest using this method.
In this technical note, we have demonstrated a proteolytic method
allowing for the evaluation of O-GlcNAcylation of proteins of interest
that are typically obscured by heavy chain during immunoprecipitation.
To our knowledge, this is the only modified immunoprecipitation pro­
cedure that allows for evaluation of the O-GlcNAcylation status of these
proteins as well as the presence of a protein of interest and any inter­
acting proteins. Potentially, this method will allow more rapid identi­
fication of transcription factors and other proteins regulated by O-
GlcNAcylation, advancing our understanding of this unique modifica­
tion’s effect on cellular processes.
Author Contributions
Miranda Machacek: Conceptualization; Investigation; Methodology;
Validation; Writing - original draft. Patrick Fields: Project administra­
tion; Resources; Funding acquisition; Writing - review & editing. Chad
Slawson: Project administration; Resources; Funding acquisition;
Writing - review & editing.
M. Machacek et al.
Declaration of competing interest
The authors declare no competing interests.
Acknowledgements
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases grant R01DK091277 awarded to P. F.,
National Institute of Diabetes and Digestive and Kidney Diseases grant
R01DK100595 awarded to C.S, the Molecular Regulation of Cell
Development and Differentiation COBRE P30GM122731 awarded to P.
F. and C. S., and a KUMC Center Biomedical Research Training Program
grant awarded to M. M.
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