Searching for protein biomarkers in Saliva

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Ivan Delgado
Ivan Delgado's picture
Searching for protein biomarkers in Saliva

 
We all appreciate that saliva is a great source of DNA for molecular work in humans. Yet, it could also be a great source of protein in studies looking for protein biomarkers, if it wasn't for the fact that saliva is loaded by amylase. A group recently developed a protocol (Electrophoresis 20:4150-4157, 2008) were they found a way to get rid of almost all amylase from saliva. They basically filled a 1-ml syringe with potato starch, pushed the saliva through this syringe, and bingo: amylase-free saliva. 
Pretty cool.

Arvind Singh Pundir
Arvind Singh Pundir's picture
Hi Ivan

Hi Ivan
is there any way that users who dont have access to this journal can have a insight to this paper, as in my case our instt. has no access to this journal

Ivan Delgado
Ivan Delgado's picture
Hi pundir,

Hi pundir,
I wished I could give a positive answer to your question but unfortunately I do not know how you would obtain a copy of this article if you did not have access to it at your institution. When I need an article and I have no direct access to the journal I typically send an email to the author and more often than not they send you a pdf. 
 
pundir wrote:

Hi Ivan
is there any way that users who dont have access to this journal can have a insight to this paper, as in my case our instt. has no access to this journal
g a
g a's picture
I CAN SEND YOU THE ARTICLE ..

I CAN SEND YOU THE ARTICLE ...... SEND ME YOUR EMAIL ADDRESS...
CHEERS...
GAGANJOT SINGH

g a
g a's picture
HERE IS THE MATERIAL AND

HERE IS THE MATERIAL AND METHOD SECTION
2 Materials and methods
2.1 Whole saliva collection
The complete saliva accumulation protocol was approved by
the Ethical Committee of Hadassah Medical Center, Jerusa-
lem, Israel. Unstimulated whole saliva ?ow was collected from
four healthy volunteers into a pre-calibrated tube using the
spitting method. Saliva samples were immediately kept on ice
and thereafter were centrifuged at 14 000g for 20min at 41Cto
remove insoluble materials, cell debris and food remnants.
The supernatant of each sample was collected and protein
concentration was determined using the Bio-Rad protein
assay according to Bradford [20] (Bio-Rad, Hercules, CA,
USA). For 2-DE, supernatants (before and after alpha amylase
removal) were frozen at 801C and lyophilized overnight.
Sediments were dissolved in 7M urea, 2M thiourea and
4%w/vCHAPSandstoredat 801C until analysis.
2.2 Alpha amylase af?nity removal
Whole saliva was subjected to the amylase removing device
(Fig. 1, provisional patent application 60915204). The device was
composed of a 1mL plastic syringe with a 0.45 mm ?lter at the
tip (Whatman FP 30/0.45). The syringe was ?lled with 1000mg
potato starch (Sigma S2630); 600 mL of water was hand pressed
(20 s) through the device to moisturize the substrate. Thereafter,
1mL of whole saliva (in two aliquots of 500 mL) was hand
pressed and ?ltrated (120 s) through the syringe. The resultant
1mL ?ltrate ?uid was an amylase-free substance.
2.3 Amylase activity
Amylase activity was determined using 96mM 3,5-Dinitro-
salicilic acid solution (Sigma D-0550) as previously
described [21, Sigma product information A0521].
2.4 Captured alpha amylase elution
After ?ltrating whole saliva as described in Section 2.3
(resulting in amylase–starch complexes), amylase was eluted
from the potato starch matrix by the addition of 1mL of
10%w/v SDS 3%v/v mercaptoethanol solution for 10min at
room temperature and vortexed for 10 s. After centrifugation
supernatant containing alpha amylase was collected for
further experiments.
Saliva sample
Potato starch
Filter
Collecting vial
2.5 SDS-PAGE analysis
Whole saliva, saliva depleted of alpha amylase and
eluted amylase (10 mg protein each) were run on an 8%
acrylamide minigel (Bio-Rad) for 70min at 110 V with
Tris-glycine-SDS buffer. Gels were stained with 0.25%w/v
coomassie Brilliant Blue for 2 h, followed by distaining
with 20%v/v ethanol/7%v/v acetic acid for 2 h. Gel images
were obtained with a GS-800 calibrated densitometer
(Bio-Rad) and the protein bands optical density were
calculated by Tina 2 software (Raytest, Straubenhardt,
Germany).
2.6 2-DE
For analytical gels, samples of 45 mg protein were subjected to re-
hydration followed by isoelectrofocusing in 18 cm long, pH
3–10 NL Immobiline DryStrips gels (Amersham Biosciences,
Uppsala, Sweden) as previously described [22–24]. Re-hydration
was carried out in 7M urea, 2M thiourea, 4%w/v CHAPS,
1%w/v dithiothreitol and 0.5%v/v carrier ampholytes (bio-lyte
40% 3/10 Bio-Rad) and a trace of bromophenol blue for 16 h,
with a constant voltage of 50 V at 201C using a Protean IEF Cell
(Bio-Rad). The voltage was then gradually increased to 10 000 V
at 201C and samples were focused for an additional 8 h. To
prepare the gel strips for separation in the second dimension the
stripsweresoakedtwicefor15mininanSDS-PAGE
equilibration buffer (6M urea, 30%v/v glycerol, 2%w/v SDS,
0.05M Tris-HCl pH 6.8, 2%w/v dithiothreitol) and then (second
equilibration) in an SDS-PAGE equilibration buffer solution but
the dithiothreitol reagent was substituted by 2.5%w/v iodoace-
tamide. For the second dimension, strips were embedded in a
0.5%w/v agarose containing a trace of bromophenol blue and
loaded onto hinged spacer plates (20 cm20.5 cm; Bio-Rad)
using a 9.5–16.5% SDS polyacrylamide gradient gel electro-
phoresis was conducted simultaneously for all samples on the
same running and staining apparatus at a constant current of
30mA per gel at 101Cuntilthebromophenolbluedyefront
band reached the bottom of the gel. For protein detection, the
gels were stained with ammoniacal silver nitrate using the
DodecaStainerShaker(Bio-Rad).
Figure 1. Illustration of alpha amylase removing device.
2.5 SDS-PAGE analysis
Whole saliva, saliva depleted of alpha amylase and
eluted amylase (10 mg protein each) were run on an 8%
acrylamide minigel (Bio-Rad) for 70min at 110 V with
Tris-glycine-SDS buffer. Gels were stained with 0.25%w/v
coomassie Brilliant Blue for 2 h, followed by distaining
with 20%v/v ethanol/7%v/v acetic acid for 2 h. Gel images
were obtained with a GS-800 calibrated densitometer
(Bio-Rad) and the protein bands optical density were
calculated by Tina 2 software (Raytest, Straubenhardt,
Germany).
2.6 2-DE
For analytical gels, samples of 45 mg protein were subjected to re-
hydration followed by isoelectrofocusing in 18 cm long, pH
3–10 NL Immobiline DryStrips gels (Amersham Biosciences,
Uppsala, Sweden) as previously described [22–24]. Re-hydration
was carried out in 7M urea, 2M thiourea, 4%w/v CHAPS,
1%w/v dithiothreitol and 0.5%v/v carrier ampholytes (bio-lyte
40% 3/10 Bio-Rad) and a trace of bromophenol blue for 16 h,
with a constant voltage of 50 V at 201C using a Protean IEF Cell
(Bio-Rad). The voltage was then gradually increased to 10 000 V
at 201C and samples were focused for an additional 8 h. To
prepare the gel strips for separation in the second dimension the
stripsweresoakedtwicefor15mininanSDS-PAGE
equilibration buffer (6M urea, 30%v/v glycerol, 2%w/v SDS,
0.05M Tris-HCl pH 6.8, 2%w/v dithiothreitol) and then (second
equilibration) in an SDS-PAGE equilibration buffer solution but
the dithiothreitol reagent was substituted by 2.5%w/v iodoace-
tamide. For the second dimension, strips were embedded in a
0.5%w/v agarose containing a trace of bromophenol blue and
loaded onto hinged spacer plates (20 cm20.5 cm; Bio-Rad)
using a 9.5–16.5% SDS polyacrylamide gradient gel electro-
phoresis was conducted simultaneously for all samples on the
same running and staining apparatus at a constant current of
30mA per gel at 101Cuntilthebromophenolbluedyefront
band reached the bottom of the gel. For protein detection, the
gels were stained with ammoniacal silver nitrate using the
DodecaStainerShaker(Bio-Rad).
HOPE THAT WORKS.....