http://www.mitosciences.com
PRODUCTS FOR RESEARCH INTO THE FUNCTION AND DYSFUNCTION OF
MITOCHONDRIAL PYRUVATE DEHYDROGENASE (PDH)
MSP01 PDH IMMUNOCAPTURE KIT
Isolates mitochondrial PDH from human and bovine tissues and cell lines.
RESEARCH USES
The pyruvate dehydrogenase complex (PDH) immunocapture
kit allows isolation of PDH from small amounts of tissue.
This facilitates the subsequent analysis of the assembly state,
activity and post-translational modification of the complex.
Pyruvate dehydrogenase is a key enzyme in energy
metabolism. It converts pyruvate that has been transported
into the mitochondrion into the 2-carbon molecule acetyl-CoA
for subsequent breakdown in the Krebs cycle (citric acid
cycle) (1). PDH activity is under tight control through
phosphorylation and dephosphorylation of the E1subunit,
reactions which involve a set of specific pyruvate
dehydrogenase kinases (PDK1-4) and phosphatases
respectively (2,3). The immunocapture antibody isolates a
functionally active complex (4) and a protocol has been
described for measuring this activity while the enzyme is still
bound to the capture beads or fixed in a 96 well plate (4).
Tightly bound kinases and phosphatases are retained on the
complex through the immunocapture step. Release of the
protein and subsequent gel electrophoresis allows
characterization of the phosphorylation state, which can then
be analyzed in more detail by mass spectrometry. Uses for
this PDH immunocapture kit include but are not limited to
examining alterations in assembly of the complex in genetic
PDH deficiency (5,6) altered PDH functioning in late-onset
diabetes (NIDMM) (7,8) and the response of the enzyme
complex to diet (9), exercise (10) and sepsis (11).
1mg mitochondria in 0.19 ml Buffer A,
1 g/ml pepstatin, 1g/ml leupeptin, 1mM PMSF

Add 10 l 10% LM incubate on ice 30 mins

Centrifuge 4000g 4oC 10 mins.

Incubate supernatant with 10 l capture resin MSP01
3 hours at room temperature OR overnight 4oC

Beads collected by gentle centrifugation (10 sec 500g) and
washed for 5 mins in PBS, 0.05 % LM, repeat

PDH eluted from the resin by addition of
50 l 0.1 M glycine, pH 2.5, 0.05 % LM
or 50 l 1 % SDS

DESCRIPTION
The key component of the PDH immunocapture kit is a
monoclonal antibody able to selectively immunocapture the
enzyme complex. The mAb is already covalently cross linked
to Protein G-Agarose for convenience of use. This material is
provided in batches of 25 l beads which have been charged
with approximately 250 g of antibody respectively. When
used as described in the protocol in Figure 1, 10 l of beads
are able to immunocapture approximately 5 g of PDH from
heart mitochondria. Also provided are 2 mg of bovine heart
mitochondria as a positive control to be used prior to, or
during, isolation of PDH from experimental samples. As an
alternative, researchers can purchase the individual
components i.e. 100 g of mAb, and 2 mg BHM (kit
MSP01c).
Protein concentration determined.
(e.g. by micro-BCA method (Pierce))

Buffer A : 50 mM Tris.HCl pH 7.5
Leupeptin : 1 mg/ml (water)
Pepstatin : 1 mg/ml (ethanol)
PMSF : 0.3 M (ethanol)
LM : n-dodecyl-b-D-maltoside
Figure 1. Example of PDH isolation procedure
from heart mitochondria
MitoScience LLC, 1850 Millrace Drive, Suite 3A, Eugene, OR 97403, +1 (800) 910-6486, (541) 284-1800, Fax (541) 284-1801
Page 1 of 2
http://www.mitosciences.com
PRODUCTS FOR RESEARCH INTO THE FUNCTION AND DYSFUNCTION OF MITOCHONDRIAL PDH
SUGGESTED PROTOCOL FOR IMMUNO-CAPTURING
PDH.
The amount of PDH that is captured in any experiment
depends on both the amount of capture antibody and the
amount of cell extract or isolated mitochondria used.
Calculation of the amount of beads to be used in any
experiment must also take account of the source of the
material from which PDH is to be isolated because
mitochondria from different tissues have different
concentrations of the enzyme complex. For example the
levels of PDH in mitochondria from cell culture material are
around 10 fold less that in heart mitochondria (12). Figure 1
shows a schematic of a generic protocol developed for
isolating PDH from heart tissue.
MATERIALS AND STORAGE
Kit MSP01 contains the anti-PDH immunocapture mAb
covalently linked to protein G-agarose beads in 25l amounts.
The beads have between 8-10 g mAb bound per l bead
volume. All volumes of bead resin are suspended in 400 l of
PBS buffer (1.4 mM KH2PO4, 8 mM Na2HPO4, 140 mM
NaCl, 2.7 mM KCl, pH 7.3) with 0.02 % sodium azide. Also
included are 2 mg of purified bovine heart mitochondria
resuspended in 400 l of heart mitochondria resuspension
buffer (10 mM Tris.HCl, pH 7.8, 0.2 M sucrose, 0.2 mM
EDTA, 1 mM PMSF). The antibody is shipped on ice. Upon
receipt store the mAb at 4oC. The mitochondrial preparation
should be aliquoted and stored at -20oC until use.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
J Nutr Sci Vitaminol (1992), 46-51
Faseb J (1990) 4, 3224-3233
Biochem J (1999) 344 Pt 1, 47-53
Anal Biochem (2003) 314, 121-127
Hum Mutat (2000) 15, 209-219
Hum Mol Genet (1997) 6, 1361-1367
Biochem J (2002) 366, 839-846
Biochem J (1984) 219, 635-646
Diabetes (2003) 52, 1371-1376
Metabolism (1999) 48, 865-869
Shock (1996) 6, 89-94
FEBS Lett (2002) 529, 173-178
Price List
 MSP01c contains 100 g of PDH immunocapture antibody plus 2 mg of
BHM control . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . .. $270
 MSP01-250 Kit to immunocapture ~12 g of PDH from heart
mitochondria plus 2 mg of BHM control . . . . . . . . . . . . . . . . . . . . .$295
All MitoSciences products are sold “FOR RESEARCH
PURPOSES ONLY”
Figure 2. Immunocapture of
PDH from bovine heart
mitochondria. Using 10 l of
capture resin MSP01, the yield
of PDH from 1000 g of bovine
heart mitochondria (BHM) is
approximately 5 g. However a
yield detectable by Coomassie
staining can be obtained from as
little as 200 g of total
mitochondria.
This yield is
enough
for
most
mass
spectrometry applications. This
mAb also captures PDH
efficiently from human sources.
MitoScience LLC, 1850 Millrace Drive, Suite 3A, Eugene, OR 97403, +1 (800) 910-6486, (541) 284-1800, Fax (541) 284-1801
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