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Detection of nonhuman primate gonadotropins in polyacrylamide gels An alternative to the western blot.

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American Journal of Primatology 26:155-166 (1992)
Detection of Nonhuman Primate Gonadotropins in
Polyacrylamide Gels:
An Alternative to the Western Blot
Wisconsin Regional Primate Research Center, Uniuersity of Wisconsin, Madison
The lack of appropriate antibodies restricts the use of western blots in
studies of nonhuman primate gonadotropins. We now present the evaluation of an alternative method that can be applied in situations in which
antibody is limiting or nonexistent. The use of nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer
(Swack et al., 1987) retains a significant degree of a-p subunit association,
as determined by the western blot analysis of human and rhesus luteinizing hormone (LH; 33 and 36 kD, respectively). This observation was
confirmed when eluates of gel slices were analyzed by specific gonadotropin immuno- and bioassays. The molecular weight of rhesus LH in pituitary protein was found to be 36 kD by bioassay and immunoassay. Similarly, the molecular weight of rhesus follicle-stimulating hormone (FSH)
did not differ between assay systems (36 kD).The analysis of chorionic
gonadotropin (CGYLH activity in gel eluates of pregnant rhesus and tamarin urinary protein revealed major activity at 43 kD, with lesser bands
between 34 and 39 kD. Guanidine-induced dissociation of gonadotropin
subunits in pregnant rhesus urinary protein resulted in the disappearance
of dimer a t 43 and 36 kD and the appearance of 25 and 16 kD subunit
peaks. This alternative to the western blot is not limited to SDS-PAGE.
Native gel electrophoresis (no SDS) showed that rhesus FSH possesses a
greater negative charge than rhesus LH (Rf = 0.49 vs. 0.35). Isoelectric
focusing PAGE resolved distinct isoforms of rhesus LH and FSH (pl range
5.2-7.2 and 4.0-5.8, respectively). The electrophoretic analysis of nonhuman primate gonadotropins in impure protein samples will provide fundamental data on the comparative biochemistry of these hormones. Applications will be found in biomedical and comparative studies.
Key words: SDS-PAGE, luteinizing hormone, follicle-stimulating hormone, chorionic gonadotropin
High-resolution, technical ease, speed, and economy have made polyacrylamide gel electrophoresis (PAGE) one of the most widely used analytical methods
Received for publication October 18, 1990; accepted March 4,1991.
Address reprint requests to Dr. Robert L. Matteri, Wisconsin Regional Primate Research Center, University of Wisconsin, 1223 Capitol Court, Madison WI, 537151299,
1992 Wiley-Liss, Inc.
156 I Matteri and Ziegler
in biochemistry. Unfortunately, there has been very little use of PAGE in studies
of nonhuman primate gonadotropins. Largely, this is due to the lack of purified
nonhuman primate gonadotropins. To progress in this area of study, we must
utilize methods that can detect specific hormones in complex protein samples in
polyacrylamide gels. The western blot is a well established and powerful technique
for the detection of specific proteins following analysis by PAGE [Towbin et al.,
19791. In brief, this method couples PAGE with specific immunodetection of the
antigen of interest. Electrophoresed proteins are transferred to a highly absorbant
sheet of material such as nylon or nitrocellulose. The sheet is incubated with a
specific antibody (in relatively high concentrations), followed by steps that allow
visualization of areas containing the antibody-antigen complex. Unfortunately,
immunoreagents do not exist or are not available in sufficient quantities for western blot analyses of gonadotropins in many species of nonhuman primates. Accordingly, we know very little about the basic biochemical properties of these
hormones. The objective of this study, therefore, was to establish a method for the
detection of gonadotropins in polyacrylamide gels that could utilize antibodies
distributed for immunoassays or bioassay systems that detect gonadotropins from
a variety of species.
Such data are vital for the validation of model systems relating to the biosynthesis and biochemistry of human hormones. In addition, a fundamental knowledge of the characteristics of reproductive hormones in rare and endangered species will be essential for the development of new reagents and methods for the
detection of these hormones in physiological studies. Effective management practices are dependent on a n accurate understanding of reproductive physiology. As
global habitat declines, we must be prepared to manage the reproduction of these
animals in captivity and wildlife preserves.
Source of Gonadotropins
A reference preparation of human pituitary gonadotropins (LER 907) was
kindly provided by the National Hormone and Pituitary Program, NIDDK. A
protein extract was prepared from two rhesus monkey pituitary glands from animals euthanized for humane reasons in accordance with the guidelines set by the
American Veterinary Association’s Panel on Euthanasia. Tissue was homogenized
on ice in 2 mlO.05 M ammonium bicarbonate, containing 5 mM benzamidine HCl
and 1 mM phenylmethylsulfonyl fluoride (PMSF). The homogenate was centrifuged a t 10,OOOg for 10 min and the resulting supernatant was immediately desalted on a 1.2 x 25 cm Sephadex G-25 column eluted with 0.05 M ammonium
bicarbonate. The protein fraction was identified by absorbance at 280 nM, pooled,
and lyophilized. The final yield of lyophilized material was 14.6 mg.
Urinary Protein
Urine was collected from a rhesus monkey (Mucacu mulattu) on day 26 of
pregnancy (4ml) and from a cotton-topped tamarin (Suguinus oedipus) of day 25 of
pregnancy (1ml). These samples were desalted and lyophilized as described for the
pituitary protein. The amounts of lyophilized protein recovered from the rhesus
and tamarin samples were 900 and 249.8 pg, respectively.
Dissociation of Gonadotropin Subunits
One-half milligram of pregnant rhesus monkey urinary protein was incubated
in 250 $ 6 M guanidine HC1 for 18 h r a t room temperature to induce dissociation
SDS-PAGE of Primate Gonadotropins / 157
of gonadotropin subunits [Matteri & Papkoff, 19871. At the end of this incubation
period, the sample was desalted and lyophilized as described above.
Protein Electrophoresis: Sodium Dodecyl Sulfate-PAGE (SDS-PAGE)
Proteins were separated on the basis of molecular size in 12.5% SDS polyacrylamide gels [Laemlli, 19701 with a nonreducing sample buffer [Swack et al., 19871.
Gels were cast in slabs (140 x 140 x 0.75 mm) or tubes (60 mm long x 5 mm I.D.).
Electrophoresis was performed at a constant current of 20 mA/slab or 2.5 mA/tube,
at 4°C. The nonreducing sample buffer contained a 4:l ratio of SDS:Nonidet P40
(NP-40; Sigma Chemical Co.), which prevents streaking and permits proper band
focusing [Swack et al., 19871. A convenient, twofold concentrate (2 x ) of the buffer
was made by combining 3.75 m10.5 M Tris HC1, pH 6.8; 0.6 gm SDS; 2 ml glycerol;
150 p1 NP-40; 800 pg bromphenol blue; and water for a total volume of 15 ml.
Equal volumes of aqueous samples and the 2 x sample buffer were combined prior
to electrophoresis.
Native PAGE
Native (no SDS) PAGE was performed as previously described [Ornstein,
19641, a t a pH of 8.3. The dimensions of the tube gels and the elution of gel slices
were the same as described above. Current was kept constant at 1.5 mA/tube.
Temperature was maintained a t 4°C.
Isoelectric Focusing (IEF) PAGE
IEF PAGE was performed in tube gels (7% polyacrylamide) containing 6.25%
Pharmalyte 3-10 (Pharmacia; wide pH range ampholytes). The anode and cathode
solutions were 0.01 M iminodiacetic acid and 0.1 M ethylenediamine, respectively.
Electrophoresis was performed at 4°C. The power supply was set to deliver 1mAt
tube, with an upper limit of 500 V. After 20 min of prefocusing, the samples were
applied in 6% ampholyte/l5% sucrose and electrophoresed for 3.5 hr.
Hormone Detection in Polyacrylamide Gels: Western Blot
Upon completion of electrophoresis in SDS slab gels, western blot analysis was
performed a s described previously [Towbin et al., 19791. Proteins were electroblotted to nylon transfer membrane (Nylon 66 Plus, Hoeffer Scientific Instruments).
The blots were blocked with 1%nonfat dry milk (Carnation) for 1 h r at room
temperature with gentle agitation. For subsequent steps, a n incubation apparatus
was used that allowed each lane to be incubated independently (Deca-Probe, Hoefer). A 2 h r incubation with primary antibody {Matteri et al., 19871was followed by
visualization with second antibody-peroxidase using 4-chloronapthol a s the substrate (GAM-HRP immunoblot kit; Bio-Rad). The primary antibody was chosen
because of its availability in large quantities and high specificity for luteinizing
hormone (LH) in a wide variety of mammalian species [Matteri et al., 19871. This
antibody was raised against bovine LH and strongly recognizes p subunit that is
free or associated with the rx subunit [Matteri et al., 19871. The antibody, generously provided by Monoclonal Antibodies, Inc., (Mountain View, CA) is distributed
for research purposes by Dr. Janet Roser (Department of Animal Science, University of California, Davis, CAI.
Passive Elution of Electrophoresed Proteins
Upon completion of electrophoresis, tube gels were cut into serial 1.25 mm
discs with a piano wire tube gel slicer. The slices were placed in a n elution buffer,
which consisted of 0.01 M phosphate, 0.15 M NaC1,2 mM EDTA, and 0.05%bovine
158 I Matteri and Ziegler
serum albumin (BSA), pH 7.4. The gel slices from pituitary and urinary protein
separations were allowed to elute passively at 4°C for at least 2 days in 500 and 250
~ 1respectively.
The eluates were analyzed by specific radioimmunoassays (RIA)
or bioassays described below.
Hormone Assays
Eluates of gel slices were analyzed for concentrations of bio- and immunoactive
LH and follicle-stimulating hormone (FSH). Reference preparations, iodinationgrade hormone and antibodies were obtained from the National Hormone and
Pituitary Program (NHPP), NIDDK. Analysis of RIA data was performed by data
reduction programs on the WRPRC VAX computer by weighted least squares
regression analysis [Rodbard and Lewald, 19701. The bioassay data were analyzed
by a previously described four-parametric algorithm [De Lean et al., 19781.
Bioassay s
Analyses of LH bioactivity were performed with a mouse interstitial cell LH
bioassay [Ziegler et al., 19871. Within- and between-assay variabilities for the LH
bioassay were 6.5% and 10.6%, respectively [Rodbard, 19741. Reference preparations for assays of LH and CG were rhLH-RP1 (rhesus LH of approximately 10%
purity) and CR-119 (iodination-grade hCG), respectively. The lowest level of detectability was 0.01 ng rhLH-RP1 and 8.6 pg CR-119itube.
FSH bioactivity was assessed by FSH-stimulated estradiol secretion in cultured rat Sertoli cells [Matteri et al., 19901. A reference preparation of approximately 10% purity was used (cynFSH-RP1). Within- and between-assay variabilities were 5.6% and 8.1%, respectively. The minimum detectable level was 0.1 ng
cynFSH-RPl/culture well.
The LH RIA was performed as directed by the instructions provided with the
reagents for the monkey LH RIA from the NHPP. Within- and between-assay
variabilities were 3.1% and 5.8%,respectively. Iodination was performed by the
iodogen method [Matteri et al., 19871. The minimum detectable level was 1.25 ng
A previously described FSH RIA was utilized [Olster & Ferin, 19871, with
reagents provided by the NHPP. Within- and between-assay coefficients of variation were 3.8% and 7.8%, respectively. Antibody characterization data from the
NHPP show this antibody to be equally reactive with free p subunit and a-p dimer.
The minimum detectable level was 0.5 ng cynFSH-RPl/tube.
The presence of immunoreactive CG and CG p subunit in SDS-gel eluates of
rhesus urinary protein was determined by RIA as described previously, with the
same monoclonal antibody used for the western blot procedure described above
[Matteri et al., 19871. Highly purified human CG (CR-119) was used for reference
and iodination. Within and between assay coefficients of variation were 4.2 and
8.0, respectively. The minimum detectable level was 0.03 ng CR-llS/tube.
Fifty microgram samples of rhesus pituitary protein and human gonadotropin
reference preparation LER-907 were analyzed by SDS-PAGE/western blot as described above. A specific anti-LH monoclonal antibody was utilized [Matteri e t al.,
19871. The lanes containing rhesus and human pituitary proteins were incubated
in solutions of 48 Fg and 1.6 Fg IgG/ml, respectively. The results of this analysis
are presented in Figure 1. Rhesus and human LH demonstrate molecular weights
SDS-PAGE of Primate Gonadotropins / 159
50 KD
39 KD
27 KD
17 KD
Fig. 1. Western blot of rhLH and hLH subsequent to separation by SDS-PAGE (12.5%) under nonreducing
conditions. Rhesus LH appears at 36 kD, while the human LH in this preparation (LER 907) is somewhat
smaller (33 kD). Some immunoreactive high-molecular-weight aggregate and free p subunit can be seen in the
lane containing human pituitary protein.
of 36 and 33 kD,respectively. A band corresponding to LH p subunit a t approximately 16 kD is seen in the lane containing human pituitary protein. High-molecular-weight aggregates also can be seen in the lane with the human sample.
Fifteen micrograms of rhesus pituitary protein were applied to a tube gel and
subjected to nonreducing SDS-PAGE. Following electrophoresis, gel slices were
placed in 500 pl elution buffer for 2 days a t 4°C. Samples of eluates from this gel
were analyzed for LH and FSH bioactivity. Immunoactive gonadotropin concentrations were determined with the macaque LH and FSH RIA reagents from
NHPP. Figure 2 shows that the molecular weights of rhesus LH and FSH are both
160 / Matteri and Ziegler
Fig. 2. Presence of rhesus LH and FSH in eluates of gel slices after nonreducing SDS-PAGE. Data are presented relative to the highest hormone value found in the gel (100%).Both hormones are readily detectable by
specific bioassays or immunoassays. The relative mobility is presented i n terms of migration relative to the dye
front (Rf).
approximately 36 kD.Hormone data were expressed as percentage of the peak
value t o facilitate qualitative visual comparisons. Hormone values in the eluates
corresponding to 100% in Figure 2 were 144.3, 177.2, 15.0, and 41.3 nglml €or
bioactive LH (bLH), immunoreactive LH (iLH), bioactive FSH (bFSH), and immunoreactive FSH (iFSH), respectively.
The molecular weights of CG in urinary protein samples from the rhesus
monkey and tamarin during pregnancy are presented in Figure 3. Approximately
400 pg of each sample was analyzed by nonreducing SDS-PAGE. Eluates of gel
slices were analyzed by immunoassay [Matteri et al., 19871 for the rhesus sample.
Since we were uncertain of antibody cross reactivity, the LH bioassay was used
SDS-PAGE of Primate Gonadotropins I 161
Pregnant Tamarin
1 .o
SDS-PAGE analysis of urinary protein from pregnant rhesus and tamarin. Data are expresse relative
to the highest hormone value found in the gel (100%).The presence of CGiLH activity in the rhesus sample was
determined by RIA. Tamarin CG/LH was detected by the mouse interstitial cell LH bioassay. The relative
mobility is presented in terms of migration relative to the dye front (Rf).
with the eluates from the tamarin sample. In both cases, a major band of activity
existed a t 43 kD, with minor forms appearing between 34 and 39 kD. The highest
hormone concentrations (100%)in the eluates from the rhesus and tamarin protein
gels were 13.6 and 5.5 ng/ml, respectively (Fig. 3, relative to purified hCG).
Figure 4 shows the detection of p subunit immunoreactivity [Matteri et al.,
19871in SDS gels of guanidine-dissociated and untreated pregnant urinary protein
from the rhesus monkey. In the untreated samples, activity is observed at 43 kD,
with a secondary form a t 36 kD. The apparent molecular weights observed after
subunit dissociation are 25 and 16 kD, respectively. Hormone values corresponding to 100% (Fig. 4) in the gel slices from the untreated and guanidine-treated
proteins were 13.6 and 17.5 ng/ml, respectively, relative to purified hCG.
Ten micrograms of pituitary protein were analyzed by native (no SDS) PAGE.
The relative mobilities of rhesus LH and FSH, detected by RIA reagents from the
NHPP, are shown in Figure 5. The mobility, and therefore apparent negative
charge, of rhesus FSH is greater than that of LH (Rf = 0.49 vs. 0.35, respectively).
The highest concentrations of iLH and iFSH in the gel eluates (100%on y axis; Fig.
5) were 100.0 and 25.2 ngiml, respectively.
Figure 6 shows immunoreactive LH and FSH obtained with 15 pg of pituitary
protein subsequent to IEF-PAGE. Hormone detection was performed with reagents
from the NHPP. The isoelectric properties of rhesus LH and FSH are consistent
with the native gel data. Molecular variants (isohormones) of LH are present with
isoelectric points (pl) between 5.2 and 7.2. FSH isohormones appear in a more
acidic range between pl4.0 and 5.8. The concentrations of iLH and iFSH in eluates
corresponding to 100% (Fig. 6) were 61.7 and 59.6 ng/ml, respectively.
The reducing conditions commonly used in SDS-PAGE present several challenges to the analysis of gonadotropins. First, loss of immunoreactivity due to
162 / Matteri and Ziegler
1 .o
Fig. 4. Molecular weight analysis of rhesus CGiLH dimer and p subunits. The data are expressed relative to
the highest hormone value found in the gel (100%).Urinary protein was treated with 6 M guanidine HCl prior
to nonreducing SDS-PAGE. The relative mobility is presented in terms of migration relative to the dye front
1 .o
Fig. 5. Analysis of rhesus LH and FSH by native (no SDS) PAGE. Data are expressed relative to the highest
hormone value found in the gel (100%).The mobility, and therefore the apparent negative charge, of FSH is
greater than that of LH. The relative mobility is presented in terms of migration relative to the dye front (Rf).
alterations in epitope configuration is a common problem under reducing conditions [Swack et al., 19871. Simply omitting the reducing agent in SDS-PAGE
sample buffer does not result in acceptable resolution and/or produces overestimations of molecular weight [Swack et al., 1987; Keel & Grotjan, 19861. Second, the
gonadotropins dissociate into subunits when reduced. This will prevent the use of
antibodies that are dimer specific. In addition, free subunits could not be detected
SDS-PAGE of Primate Gonadotropins / 163
Fig. 6. Analysis of rhesus LH and FSH by IEF PAGE. Data are expressed relative to the highest hormone value
found in the gel (100%).Distinct isohormones of both gonadotropins are resolved, with FSR extending into a
more acidic range than LH.
by bioassays. Accordingly, our primary goal was to identify conditions that would
permit the accurate electrophoretic resolution of gonadotropins while maintaining
the structural conformation needed for hormone detection. Specifically, we were
interested in evaluating a nonreducing SDS-PAGE sample buffer that was developed for maintaining epitope stability of labile membrane glycoproteins [Swack et
al., 19871. This technique would permit the first comparative studies of the physical characteristics of nonhuman primate gonadotropins by high-resolution electrophoretic methods.
The western blot presented in Figure 1demonstrates a significant retention of
the a-P subunit association of rhesus and human LH with this method. The LH p
subunit seen in the lane containing human pituitary protein may represent free
subunit present in the sample or produced by the analysis. A small amount of
rhesus LH p subunit might not be visualized due to lack of detection sensitivity.
The difference in staining intensity between human and rhesus LH reflects a
difference in relative affinity between the antibody and membrane-bound antigen
between the two species. Although the use of large quantities of antibody can
overcome detection problems due to weak antigen-antibody affinities in western
blot applications, this rarely is a practical solution.
The detection of nonhuman primate gonadotropins eluted from SDS-PAGE gel
slices represents a highly sensitive, accurate method for the molecular weight
analysis of LH, FSH, and CG. Although SDS will denature proteins, gonadotropin
bioactivity and immunoactivity are both retained following nonreducing SDSPAGE. Either the denaturation is relatively mild, or renaturation occurs in the
buffer used to elute the proteins from the gel slices. Dissociated subunits of rhesus
FSH (Fig. 2) and CG (Fig. 4) were not detected under nonreducing conditions by
RIAs known to recognize the free p subunits. The retention of structural integrity
permits the detection of hormone by bioassay (Figs. 2 and 3). This provides a
unique opportunity for comparative electrophoretic analyses of gonadotropins between species. The characterization of CG from a variety of primates has been
164 / Matteri and Ziegler
performed by size-exclusion chromatography [Hobson & Wide, 19811. Molecular
weight data were not specified, however, probably due to the well known observation that glycoproteins demonstrate abnormally high molecular weights when
applied to size-exclusion matrices. For example, baboon and human CG are apparently larger than bovine serum albumin (MW = 66 kD) by Sephadex G-100 chromatography [Bambra, 19871. The molecular weight of purified human CG by SDSPAGE, however, is between 43 and 60 kD [Wang et al., 19881. To our knowledge,
comparative electrophoretic data do not exist for nonhuman primate LH, FSH,
or CG.
The analysis of nonhuman primate gonadotropin subunits by PAGE will be
essential for the structural evaluation of these molecules. Although antibodies
that bind to reduced subunits may exist, none of the reagents available to this
laboratory possessed this ability. Accordingly, we induced subunit dissociation
with guanidine HC1 [Matteri & Papkoff, 19871 prior to separation by nonreducing
SDS-PAGE. This chaotropic salt disrupts the noncovalent binding of a and p subunits. Since dissociated subunits are not bioactive, the detection of gonadotropin
subunits by this method depends on the availability of a n antibody that will interact with free subunit. We had access to such a n antibody for rhesus CG p
[Matteri et al., 19871 but not a subunit. The analysis of urinary protein from the
pregnant rhesus monkey revealed two peaks of intact hormone at 43 and 36 kD,
which shifted to 25 and 16 kD following exposure to guanidine. The 43 kD peak
undoubtedly represents CG [Wang et al., 19881. The 36 kD peak may represent LH
[Farmer & Papkoff, 19791 or a n incompletely glycosylated variant of CG [Wang et
al., 19881. Protein that had been treated to induce subunit dissociation showed a
similar CG profile to the untreated sample but demonstrated molecular weights
consistent with those known for forms of human CG p [Wang et al., 19881 or LH p
[Farmer & Papkoff, 19791.
A major objective of this study was to evaluate the efficacy of nonreducing
SDS-PAGE for the electrophoretic analysis of nonhuman primate gonadotropins. It
also should be noted that gonadotropins eluted from sections of native or IEF
polyacrylamide gels also can be detected in order to evaluate relative charge and
isoelectric properties. Immunoreactive hormone profiles are presented in Figures 5
and 6; however, bioactivity also is retained with these nondenaturing methods of
electrophoresis (not shown). Due to the higher content of sialic acid, FSH demonstrates a greater mobility (negative charge) than LH by native PAGE and a lower
isoelectric range by IEF PAGE.
The detection of eluted hormones from gel slices may provide the opportunity
for the first physical characterizations of gonadotropins from a wide variety of
primate species. Although results from only two species are given in the present
study, we have utilized this method in preliminary work with human LH and CG
(31 and 45 kD, respectively [Dumesic et al., 19901) as well as with marmoset and
tamarin LH (studies in progress). Using a n RIA for prolactin [Bethea and Papkoff,
19861, we have obtained molecular weight data on rhesus placental lactogen in
amniotic fluid protein and rhesus prolactin in pituitary protein separated by SDSPAGE (unpublished).To date, we have found the present technique to be effective
with any substance for which a suitable assay exists.
The method described herein offers a variety of attractive features: 1)Bioassays may be utilized that are not species specific. Such assays are the only means
of detection available for gonadotropins from many primate species. The excellent
detection sensitivity of in vitro gonadotropin bioassays [Ziegler et al., 1987; Matteri et al., 19901 offers the potential for hormone detection with very small
amounts of electrophoresed sample. 2) If hormone assays are performed with well
SDS-PAGE of Primate Gonadotropins / 165
characterized reference preparations, excellent quantitation can be achieved. 3)
Valuable assay reagents could be conserved, since immunoassay systems that are
less than suitable for quantitation of physiological samples due to nonparallelism
could still be used to provide accurate qualitative data. 4) Depending on hormone
content of the sample and availability of assay reagents, multiple hormones can be
detected from the same gel. Only 50% of the available gel eluate volumes were
utilized to generate the data in Figure 2. Maximizing the data obtained from each
sample may be critical due to the limited amounts of material usually available
from nonhuman primates
1. The molecular weights of gonadotropins can be determined by hormone
assay data of eluates from sections of nonreducing SDS polyacrylamide gels. Since
bioassays can be used a s well as immunoassays, this method offers great promise
for studies of gonadotropins for which antibodies are limiting or nonexistent.
2. Chaotropic salts, such as guanidine HCl, can be used to induce subunit
dissociation without the use of a reducing agent prior to nonreducing SDS-PAGE.
Subunits eluted from gel slices can be detected by RIA.
3. The relative charge and isoelectric characteristics of nonhuman primate
gonadotropins can be determined from eluates of native and IEF gels as well as
SDS gels.
We are grateful to Elaine Anderson and Ann McCormick for preparing the
manuscript. We thank Neal S. Rhutasel, Frederick Wegner, Guenther Scheffler,
and Daniel Wittwer for expert technical assistance. We also thank NIDDK and the
National Hormone and Pituitary Program (University of Maryland School of Medicine) for gifts of hormones and reagents for assay purposes. Rat testicular tissue
for the FSH bioassays was kindly provided by Mary Herman-Yost, Craig Schulz,
and Dr. Philippa Claude, Wisconsin Regional Primate Research Center, in the
course of their research with cultured rat adrenal medullary cells. This work was
supported by NIH grant RR00167 to the Wisconsin Regional Primate Research
Center. This is publication 30-032 of the Wisconsin Regional Primate Research
Center. This study was conducted in accordance with the “Guide for the Care and
Use of Laboratory Animals,” NIH Publication No. 85-23 and Public Law 89-544,
“The Animal Welfare Act,” August 24, 1966, and its Amendments.
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