Immun-Star
™
AP
Chemiluminescent Protein
Detection Systems
For Use With Nitrocellulose
and PVDF Membranes
Instruction Manual
Catalog #
170-5010, 170-5011, 170-5012,
170-5013, 170-5014, 170-5015,
170-5018, 170-5056, and 170-5057
Table of Contents
Section 1 Preparation ..........................................1
1.1 Introduction ............................................................1
1.2 Method Overview ..................................................1
1.3 Immun-Star Products ............................................2
1.4 Complementary Products ......................................3
1.5 Storage and Stability of Kit Components ................4
1.6 Safety Instructions ..................................................6
Section 2 Assay Instructions................................6
2.1 Experimental Strategy
and General Recommendations ............................6
2.2 Reagents Flowchart................................................11
2.3 Working Solutions ..................................................12
2.4 Assay Procedure ....................................................15
Section 3 Troubleshooting ..................................19
3.1 Troubleshooting Guide ..........................................19
References and Acknowledgements ......................26
Note on Electrophoresis and Blotting
Equipment..............................................................27
Appendix 1 Total Protein Detection Procedure ......28
* Note important blocking solution instructions on
page 13. High concentrations of blocker can lead to
high background on blots.
C
Section 1
Preparation
1.1 Introduction
The Immun-Star chemiluminescent detection system is a
sensitive, nonisotopic method for immunodetection of
specific antigens immobilized on nitrocellulose or PVDF
membrane. The system uses the powerful CDP-Star
chemiluminescent substrate and enhancer, which is activated
by an alkaline phosphatase enzyme conjugate. Drawing on
Bio-Rad’s expertise in protein blotting, the substrate and
enhancer are incorporated into a sensitive and easy-to-use
western blotting detection protocol.
The Immun-Star anti-mouse and Immun-Star anti-rabbit
chemiluminescent detection kits provide enough reagents to
assay 2,500 cm
2
of membrane or approximately 50 mini blots
of ~50 cm
2
each. The blotting reagents pack provides all the
complementary buffer reagents needed for western blotting,
matched in quantity to the detection kits. We also offer the
Immun-Star Intro chemiluminescent kits, with enough of all
reagents needed for 2 large blots or 8 mini blots. The
Immun-Star assay kits are for laboratory use only.
1.2 Method Overview
The first step in western blotting is the transfer of antigen onto
a solid support membrane by one of several methods. The
transfer can be done electrophoretically, following separation
1
of the antigen in a polyacrylamide or agarose gel, passively by
directly spotting the antigen onto a membrane, or by vacuum
filtration using a microfiltration apparatus. Following antigen
binding, the remaining protein binding sites on the membrane
surface are blocked with nonfat dry milk, or other protein
blocking agents.
The membrane with bound antigen is then incubated with a
primary antibody specific for the antigen to be detected. The
blot is washed to remove unbound antibody and incubated
with the respective second antibody, which has been
conjugated to alkaline phosphatase (AP). The membrane is
then treated with the chemiluminescent substrate alone for
PVDF membranes, and with substrate and enhancer for
nitrocellulose. The concentrated enhancer is added to the 1x
substrate solution and does not add any extra incubation or
wash steps. The blot is then used to expose X-ray or instant
film, or imaged by an imager capable of detecting chemiluminescent signals, such as the Bio-Rad VersaDoc
™
or ChemiDoc
™
system.
1.3 Immun-Star Products
Catalog # Description
170-5010 Immun-Star Goat Anti-Mouse -AP Detection Kit
170-5011 Immun-Star Goat Anti-Rabbit -AP Detection Kit
170-5012 Immun-Star AP Substrate Pack
170-5018 Immun-Star AP Substrate
170-5013 Immun-Star Goat Anti-Mouse -AP Intro Kit
170-5014 Immun-Star Goat Anti-Rabbit -AP Intro Kit
2
Catalog # Description
170-5015 Blotting Reagents Pack
170-5056 GAR-AP Blotting Starter Kit
170-5057 GAM-AP Blotting Starter Kit
1.4 Complementary Products
Catalog # Description
Individual Blotting Grade Reagents
170-6518 Goat Anti-Rabbit IgG (H+L)-AP, 1 ml
170-6520 Goat Anti-Mouse IgG (H+L)-AP, 1 ml
170-6521 Goat Anti-Human IgG (H+L)-AP, 1 ml
170-6435 Premixed Tris-Buffered Saline, 10x, 1 L
170-6531 Tween 20, 100 ml
170-6404 Blotting Grade Blocker, nonfat dry milk, 300 g
Blotting Membranes
Nitrocellulose Membrane, 0.45 µm
162-0113 Sheets, 20 x 20 cm, 5
162-0114 Sheets, 9.2 x 15 cm, 10
162-0115 Roll, 33 cm x 3 m, 1
162-0116 Sheets, 15 x 15 cm, 10
162-0117 Sheets, 9 x 12, 10
162-0145 Sheets, 7 x 8.4 cm, 10
162-0148 Sheets, 11.5 x 16 cm, 10
Nitrocellulose Membrane, 0.2 µm
162-0112 Roll, 33 cm x 3 m, 1
162-0146 Sheets, 7 x 8.4 cm, 10
162-0147 Sheets, 13.5 x 16.5 cm, 10
Immun-Blot PVDF Membranes
162-0175 Sheets, 10 x 15 cm, 10
3
Catalog # Description
162-0176 Sheets, 20 x 20 cm, 10
162-0177 Roll, 26 cm x 3.3 m, 1
162-0174 Sheets, 7 x 8.4 cm, 10
Blotting Standards
161-0306 Biotinylated Standards, low range, 250 µl
161-0308 Biotinylated Standards Kit (AP), low range
161-0311 Biotinylated Standards, high range, 250 µl
161-0313 Biotinylated Standards Kit (AP), high range
161-0319 Biotinylated Standards, broad range, 250 µl
161-0322 Biotinylated Standards Kit (AP), broad range
161-0363 Precision Plus Protein™ Unstained Standards
161-0382 Precision Protein™ StrepTactin-AP conjugate
170-6533 Avidin-AP, 1 ml
161-0324 Kaleidoscope™ Prestained Standards
161-0325 Kaleidoscope Polypeptide Standards
161-0375 Precision Plus Protein Kaleidoscope Standards
161-0305 Prestained SDS-PAGE Standards, low range
161-0309 Prestained SDS-PAGE Standards, high range
161-0318 Prestained SDS-PAGE Standards, broad range
1.5 Storage and Stability of Components
Quantity Shelf
Description Provided Storage Life
Catalog #170-5010/170-5011
Immun-Star Detection Kits
– 2,500 cm
2
• Immun-Star chemiluminescent substrate 125 ml 4°C 1 yr
• Immun-Star enhancer** 6.25 ml 4°C 1 yr
• AP conjugated antibody* 0.5 ml -20°C 1 yr
4
Quantity Shelf
Description Provided Storage Life
Catalog #170-5012
Immun-Star Substrate Pack
– 2,500 cm
2
• Immun-Star chemiluminescent substrate 125 ml 4°C 1 yr
• Immun-Star enhancer** 6.25 ml 4°C 1 yr
Catalog #170-5018
Immun-Star Substrate
– 2,500 cm
2
• Immun-Star substrate 125 ml 4°C 1 yr
Catalog #170-5013/170-5014
Immun-Star Introduction Kits
– 8 mini blots
• Immun-Star chemiluminescent substrate 20 ml 4°C 1 yr
• Immun-Star enhancer** 1 ml 4°C 1 yr
• AP conjugated antibody* 0.1 ml -20°C 1 yr
• TBS (10x) 220 ml 4°C or RT 1 yr
• Nonfat dry milk 2 g 4°C or RT 1 yr
• Tween 20 2.5 ml 4°C or RT 1 yr
Catalog #170-5015
Blotting Reagents Pack
– 2,500 cm
2
• 10x TBS 2 x 1 L RT 1 yr
• Tween 20 15 ml RT 1 yr
• Nonfat dry milk blocker 10 g RT 1 yr
* Store conjugate at -20°C until first use, then aliquot and store at -20°C. Avoid
repeat freeze/thaw cycles. 4°C storage is also acceptable.
**Immun-Star enhancer is used with nitrocellulose membrane only.
5
1.6 Safety Instructions
1. Read the entire instruction manual before beginning the
assay.
2. Wear gloves and protective clothing, such as a laboratory
coat and goggles, when preparing and working with the
solutions in the assay. The Immun-Star enhancer contains
diethanolamine, which can cause skin and eye irritation. In
case of contact, immediately flush the skin or eyes with
copious amounts of water for at least 15 min, and remove
contaminated clothing.
Note: See Material Data Safety Sheet on diethanolamine
for additional information.
3. Work in well-ventilated areas. Avoid inhalation of vapors
when handling solutions containing diethanolamine.
4. Do not mouth-pipet any solutions.
Section 2
Assay Instructions
2.1 Experimental Strategy and General
Recommendations
Temperature. All steps are performed at room temperature
(22–25°C) unless indicated otherwise in the instructions. If a
lower assay temperature is preferred, it is advisable to double
the incubation and wash times for each 10°C decrease in
temperature.
6
Water Purity. Use only deionized distilled water to prepare
all solutions. In addition, care should be taken to prevent
alkaline phosphatase contamination of assay solutions.
Ideally, distilled deionized water (ddH
2
O) should be autoclaved
or sterile-filtered prior to use in buffers and solutions.
Membrane Selection. The Immun-Star assay is specially
designed for use with nitrocellulose and PVDF membranes.
This kit is not compatible with nylon membrane. The use of
Immun-Star enhancer is required when performing blots on
nitrocellulose membrane. Longer exposure times will be
necessary if the enhancer is not used, with resulting interference from background development. If proteins are
transferred to PVDF membrane, use of the enhancer is not
necessary. All other steps in the assay procedure remain the
same.
Primary Antibody. Generally, when serum or tissue culture
supernatants are the source of primary antibody, a
1:100–1:1,000 dilution of the primary antibody in buffer is
used for detection of antigens on the membrane surface. For
chromatographically purified monospecific antibodies, a
1:500–1:10,000 dilution in buffer is used for antigen detection.
A 1:1,000–1:100,000 dilution is used when ascites fluid is the
source of antibody. Optimal dilution factors must be determined experimentally. The optimal antibody concentration is
usually considered to be the greatest dilution of antibody
reagent that still results in a strong positive signal without
significant membrane background or nonspecific reactions.
7
Blotting Grade Conjugates. The conjugates supplied by
Bio-Rad should be used in the concentrations indicated in
Section 2.3. Using a conjugate at higher concentrations may
result in an overall increase in background without any increase
in detection sensitivity.
Washes and Incubations. Continuous gentle agitation
should be used during all reactions. For best results, an orbital
shaker should be employed to maintain a uniform exposure of
the membrane to the solution.
Detergents. Tween 20 is essential in washing to eliminate
overall background and nonspecific hydrophobic reactions. At
0.05%–0.1%, Tween 20 will not disrupt binding of primary
antibodies to antigens or antigens to the membrane, but will
optimize detection sensitivity by eliminating nonspecific
reactions. Increased concentrations of Tween 20 (up to 0.3%)
can be used if background problems persist. Alternative
detergents should not be substituted. The wash between the
blocking step and incubation with the first antibody is
essential and should not be altered.
Molecular Weight Standards. Biotinylated SDS-PAGE
standards and Precision Plus Protein
™
unstained standards are
recommended for molecular weight determinations with the
Immun-Star assays. The Biotinylated SDS-PAGE standards are
detected by binding avidin-AP or streptavidin-AP to the biotinylated proteins, which will produce a chemiluminescent signal
upon reaction with the substrate. The Precision Plus Protein
8
9
unstained standards are detected by incubating with
StrepTactin-AP. Because the avidin-AP (or streptavidin-AP) and
StrepTactin-AP can be directly added to the second antibody
solution, no extra steps are needed to detect the biotinylated or
Precision Plus Protein unstained standards. The standards
should be diluted 1:50 in electrophoresis sample buffer (note:
this is a higher dilution than recommended in the biotinylated
standards manual). Load 10 µl per lane for mini gels and 15 µl
per lane for full size gels.
Prestained SDS-PAGE and Kaleidoscope prestained
standards can also be used for assessing the transfer
efficiency of samples.
Total Protein Detection. In the identification of specific
antigens, total protein staining is required to correlate the
signal detected to a complex protein mixture. Any of the
common total protein staining methods will work to give a
profile of the blot, including colloidal gold, Amido Black, and
Ponceau S. If you want to generate a chemiluminescent total
protein blot, there are Biotin Blot
™
systems that allow
visualization by chemiluminescent signal. The chemiluminescent detection method uses NHS-biotin to biotinylate all the
proteins on the membrane. The biotinylated proteins are
detected with avidin-AP or streptavidin-AP and visualized with
the chemiluminescent substrate (see Appendix 1 for details).
2.2 Reagent Flowchart
Several of the solutions you will make are end-use solutions
and are also used to make up other solutions. For example,
1x TBS is used as a wash solution, for the blocking solution,
and to make TTBS. In turn, TTBS is used both as a wash
solution (end use), and as part of the antibody buffer.
This reagent flowchart shows the overall picture of the use of
some of the reagents. Since there are many ways to go about
making solutions, this flowchart can help you make best use
of your time and reagents. The straightforward solutions were
left out of the flowchart.
Refer to Section 2.3, Working Solutions, for the exact
quantities and contents of all the solutions.
10
11
Reagent Flowchart. (Does not include all reagents
in this kit.)
10x TBS
stock
Nonfat
dry milk
Nonfat
dry milk
End-use
wash solution
1x TBS
Tween 20 TTBS
TTBS
End-use wash solution
Blocking solution
Antibody buffer
+
+
+
12
2.3 Working Solutions (based on 200 cm2of
membrane; one large blot, or four mini gel blots)
This kit has been designed to blot 2,500 cm
2
of membrane, or
approximately 50 mini blots. The Working Solutions section of
this manual has been based on enough of each solution to
blot 200 cm
2
of membrane at 0.5 ml of solution per cm2for
each step, except the substrate/enhancer step, which
requires only 0.05 ml per cm
2
. For practical reference,
200 cm
2
of membrane is approximately the size of one large
western blot, or four mini gel western blots. If a larger or
smaller number of blots is used, vary the quantity of reagents
by the appropriate amount.
Since any blotting procedure is best when it is performed with
fresh solutions, it is preferable to make up the solutions
needed for the day’s blots. If solutions containing proteins are
made and held over a period of days, they must be refrigerated. On the day that the solutions are going to be used, they
should be warmed to room temperature before use to avoid
adding any new variables to the blotting procedure.
Tips
It is important that enough reagent be used to cover the entire
blot. In order to make most efficient use of the reagents, the
container used for wash and incubation steps should fit the
blot as closely as possible.
The enhancer used in this procedure is difficult to wash from
containers, so care should be taken to use disposable or
13
dedicated-use containers to avoid contaminating other
experiments.
Because of the high sensitivity of the chemiluminescent assay,
care should be taken to avoid alkaline phosphatase contamination of the buffers and solutions used in the procedure. For
best results, the ddH
2
O used to prepare solutions should be
autoclaved or filtered prior to use in this assay.
Tris-Buffered Saline (1x TBS)
(20 mM Tris, 500 mM NaCl, pH 7.5)
If using Bio-Rad liquid concentrate 10x TBS, add 110 ml of
10x TBS to 990 ml of ddH
2
O. Label this bottle “1x TBS”.
If using solid TBS powder, follow instructions to make 1,100
ml of a 1x TBS solution. Typically, a 1x solution is 32.3 g of
TBS brought up to 1 L of solution with ddH
2
O.
Wash Solution (TTBS)
(20 mM Tris, 500 mM NaCl, 0.1% Tween 20, pH 7.5)
Add 0.8 ml of Tween 20 to 800 ml of 1x TBS. Label this bottle
“TTBS”.
Blocking Solution
(0.2% nonfat dry milk in TBS)
Add 0.2 g of nonfat dry milk to 100 ml of 1x TBS. Stir until
dissolved. Label this solution “Blocking Solution”.
Suggestions for other blockers:
casein – 0.2% in TBS, gelatin – 3% in TBS or BSA or 3% in TBS
Antibody Buffer
(0.2% nonfat dry milk in TTBS)
Add 0.4 g of nonfat dry milk to 200 ml TTBS. Stir until
dissolved. Label this solution “Antibody Buffer”.
Primary Antibody Solution
Dilute the primary antibody to the appropriate titer in 100 ml of
antibody buffer. Label this solution “Primary Antibody
Solution”.
Second Antibody Conjugate Solution (1:3,000)
Add 33 µl of the second antibody conjugate to 100 ml of
antibody buffer. Label this solution “Second Antibody
Solution”.
Substrate Solution for PVDF membrane blots
Immun-Star generates a very fast light signal on PVDF
membrane; therefore, the use of the enhancer is not
necessary. Use 10 ml of chemiluminescent substrate per
200 cm
2
. The substrate is provided ready to use.
Substrate Solution for nitrocellulose membrane blots
Add 500 µl of the enhancer reagent to 10 ml of Immun-Star
chemiluminescent substrate. Label this solution “Substrate
Solution”. This solution can be stored at 4°C for up to 1 week.
14
2.4 Detailed Assay Procedure
Note: Before beginning, read through the entire procedure.
1. Antigen application – apply antigen to the membrane
surface using one of the three basic methods described
below. A small amount of known antigen or primary
antibody dotted on one corner of the membrane prior to
blocking will produce a positive reaction if the procedure is
successful.
a. Electrophoretic blotting – the antigens of interest
are electrophoretically transferred to the membrane from
a gel (i.e., SDS-PAGE gel, IEF gel, or native gel) using
the Trans-Blot
®
cell, Mini Trans-Blot®cell, Criterion
®
blotter, or Trans-Blot SD cell. If desired, cut the wet
membrane into 0.6–0.8 cm wide strips. Immerse the
strips or the entire sheet in TBS before proceeding to
the blocking step.
b. Microfiltration blotting – the Immun-Star assay can
easily be adapted for use in the Bio-Dot
®
and Bio-Dot
SF apparatus. These instruments allow rapid,
reproducible applications of up to 96 samples on one
membrane sheet. The membrane should be removed
from the apparatus after antigen application. Because
nonfat dry milk cannot be filtered through the
membrane in the Bio-Dot or Bio-Dot SF apparatus, the
blocking and incubation steps should be carried out in
a separate container.
15
c. Dot-blotting – cut the membrane sheet to the
appropriate size. Draw a grid on the membrane with a
pencil. Wet the dry membrane by slowly sliding the
membrane at a 45° angle into TBS. (PVDF membranes
must first be wetted in methanol; consult membrane
instructions for complete information.) Remove the
thoroughly wetted membrane from the TBS and dry it
on filter paper for approximately 5 min. Apply antigen
sample to each grid square using a syringe or a
variable pipet, by displacing a 1 µl drop of sample to
the tip of the syringe or pipet and gently touching it to
the surface of the membrane. If the sample is very
dilute, it is possible to apply successive 1 µl doses at
the same spot by letting the previous sample application dry completely before adding an additional dose.
In all cases, the membrane should be allowed to dry
completely before proceeding to the blocking step. For
PVDF following this drying step, rewet with 50%
methanol before transferring to the TBS solution in step
2 below.
2. Wash – following transfer, wash the membrane in TBS for
5–10 min with gentle agitation at room temperature (RT).
Decant the wash solution and repeat the wash step one
more time. These washes are important to reduce spotted
or blotchy background problems.
3. Blocking step – after the antigen is applied, using one of
the above methods, immerse the membrane, at a
16
45° angle, into the blocking solution. Gently agitate the
solution using an orbital shaker platform and incubate for
30 min to 1 hr at RT, or block overnight at 4°C.
4. Wash – decant the blocking solution and add TTBS to the
membrane. Wash for 5–10 min with gentle agitation at RT.
5. Primary antibody incubation – decant the TTBS and
add the primary antibody solution to the membrane.
Incubate 1–2 hr with gentle agitation at RT. Overnight
incubation may be preferred, since longer incubation
periods may increase the sensitivity of detection. The
optimum conditions of dilution and incubation time must
be determined experimentally. For overnight incubations,
4°C is recommended.
6. Wash – decant the primary antibody solution and add
TTBS to the membrane. Wash for 5–10 min with gentle
agitation at RT. Decant the wash solution and repeat the
wash step, with additional TTBS.
7. Second antibody conjugate incubation – decant the
TTBS and add the second antibody solution. Incubate for
30 min to 2 hr using gentle agitation at RT.
8. Wash – decant the conjugate solution and add TTBS to
the membrane. Wash for 5–10 min with gentle agitation at
RT. Decant the wash solution and repeat the wash step
two more times with additional TTBS.
17
9. Blot development – remove the membrane and drain
excess liquid without letting the blot dry. Place on a piece of
Saran wrap on a level surface. Pipet the substrate
solution onto the membrane. Incubate 5 min. Remove the
membrane, draining the excess liquid from the blot. Seal in
a heat-sealable bag or in Saran wrap. Do not allow the
membrane to dry, because this will result in a loss of signal.
10. Film exposure/imaging – expose X-ray or instant film
to the blot at RT. Exposure time will depend on the type of
membrane used and the protein concentration. Table 1
lists recommended exposure times; however, optimal
conditions should be determined for each particular application. Develop the film according to the manufacturer’s
instructions. Alternatively, a Bio-Rad imager such as the
Bio-Rad VersaDoc
™
or ChemiDoc™system can be used
to capture the signal.
Table 1. Typical exposure times for nitrocellulose
and PVDF membranes.
With Immun-Star Without
Membrane Enhancer Enhancer
Nitrocellulose 1–10 min 12–24 hr
PVDF Not recommended 1–10 min
18
Section 3
Troubleshooting
3.1 Troubleshooting Guide
Probable Recommended
Problem Cause Solution
1. No reaction or weak a. Exposure time was i. Increase the
signal. too short. exposure time.
b. Immun-Star enhancer i. Use the
was not used with Immun-Star
nitrocellulose enhancer assay
membrane. protocol.
Exposure times
will vary; for
example, a
nitrocellulose
blot without
enhancer would
require a much
longer exposure
time to generate
a signal
comparable to
that generated
with enhancer in
5 min.
19
Troubleshooting Guide (continued)
Probable Recommended
Problem Cause Solution
c. Blot was allowed to i. Use heat-
dry after incubation sealable
with the chemilumi- bags to prevent
nescent substrate. drying of the
membrane.
d. Chemiluminescent i. Store the
substrate solution is reagent
inactive. at the proper
temperature,
4°C.
To test activity of
substrate, mix
1 ml of substrate
with 1–2 µl of
undiluted
conjugate and
look for light
emission in the
darkroom.
e. Primary antibody i. Store the anti-
solution is inactive or body solution at
nonsaturating. the proper
temperature.
Avoid bacterial
contamination,
heat inactivation,
and repeated
freeze-thaw
cycles.
20
Troubleshooting Guide (continued)
Probable Recommended
Problem Cause Solution
ii. Antibody titer
was too low.
Increase the
concentration of
the antibody
used in the
assay.
iii. Tween 20 may
affect the
reactivity of some
antibodies.
Eliminate Tween
20 from the
assay (except the
wash after the
blocking step).
f. Conjugate is inactive. i. Store the
conjugate at the
proper
temperature as
recommended in
Section 1.5.
Avoid repeated
freeze-thaw
cycles.
21
Troubleshooting Guide (continued)
Probable Recommended
Problem Cause Solution
ii. The concentra-
tion of the
conjugate was
nonsaturating.
Increase the
concentration of
the conjugate
used in the
assay.
iii. Conjugate may
be contaminated,
causing inactivation of the antibody or enzyme.
Tap water may
cause inactivation; use only
ddH
2
O to pre-
pare all solutions.
g. Little or no antigen i. Tween 20 may
is bound to the wash bound
membrane. antigen from
the membrane.
Eliminate
Tween 20 from
the assay (except
the wash after the
blocking step).
22
Troubleshooting Guide (continued)
Probable Recommended
Problem Cause Solution
ii. Transfer of
protein onto the
membrane was
incomplete. Stain
gel to assure
transfer of
protein. Use
prestained
standards to
monitor transfer
efficiency.
Consult the
appropriate
instrument
manual for
proper procedures and recommendations.
h. Primary antibody i. Loss of reactivity
is not specific or may have
does not recognize occurred during
denatured antigens electrophoretic
(common with transfer. Pretest
monoclonals). the reactivity of
the antibody
against the
antigen by a dot
blot.
23
Troubleshooting Guide (continued)
Probable Recommended
Problem Cause Solution
2. High background. a. Exposure time was i. Decrease
too long. exposure time.
ii. The use of
Immun-Star
enhancer is
required when
using nitrocellulose membranes.
This will allow
decreased film
exposure times.
b. TBS washes after i. The washes are
transfer were critical to reduce
omitted or insufficient. spotted or
blotchy
background
development.
c. Blocking was i. Increase the
insufficient. duration of the
blocking step
and/or the
concentration of
blocker used.
ii. Try a different
blocking reagent,
such as casein,
gelatin, or BSA.
24
Troubleshooting Guide (continued)
Probable Recommended
Problem Cause Solution
d. Wash stringency i. Tween 20 is
was insufficient. necessary in
wash steps to
reduce background. The
concentration
can be increased
up to 0.3% if
background
persists.
ii. Increase the
number and
length of washes.
e. Second antibody i. Use the recom-
conjugate was used mended dilution,
at an excessive con- or determine the
centration. optimal dilution
experimentally.
f. Contamination i. Refer to the
occurred during instrument
transfer. instruction
manual for recommendations.
g. Solutions and i. Autoclave or
buffers are sterile-filter
contaminated with ddH
2
O prior
alkaline to use in making
phosphatase. solutions.
25
Troubleshooting Guide (continued)
Probable Recommended
Problem Cause Solution
ii. Use blotting
grade nonfat dry
milk blocker,
which has been
quality-control
tested for
acceptable
background
levels.
iii.Avoid bacterial
contamination of
all solutions by
storing at
recommended
temperatures.
References and Acknowledgements
1. Bronstein, I., Juo, R. R., Voyta, J. C. and Edwards, B.,
Bioluminescence and Chemiluminescence: Current status, John
Wiley, Chichester, England (1991).
CDP-Star is a trademark of Tropix, Inc. Saran is a trademark of S.C.
Johnson Home Storage, Inc. StrepTactin is a trademark of Institut für
Bioanalytik GmbH. Tween is a trademark of ICI Americas, Inc.
26
Note on Electrophoresis and Blotting
Equipment
Bio-Rad provides a complete line of electrophoresis,
electrophoretic transfer, and microfiltration apparatus that can
be used with the Immun-Star assays. For more information in
the U.S. contact Bio-Rad Laboratories technical support at
1-800-4BIORAD (1-800-424-6723), or contact your local
Bio-Rad representative.
27
Appendix 1
Total Protein Detection Procedure
Required Reagents
Catalog # Description
170-6533 Avidin-AP, 1 ml
or
170-3554 Streptavidin-AP, 0.5 ml
170-6529 N-Hydroxysuccinimide Biotinate (NHS-Biotin), in
dimethylformamide, 75 mM, 4 ml
170-6435 TBS Buffer Solution, 10x, 1 L
161-0715 Tween 20, 100 ml
Additional Reagents Required
Sodium borate, 10-hydrate (Na
2B4O7
•10 H2O), ACS reagent
grade
Sodium chloride (NaCl), ACS reagent grade
Working Solutions
Borate-Tween (1x BT)
(0.05 M Na
2B4O7
•10 H2O, 0.5 M NaCl, 0.2% Tween 20,
pH 9.3)
Dissolve 38.14 g Na
2B4O7
•10 H2O and 58.44 g NaCl in 1.9 L
ddH
2
O. Add 4 ml Tween 20, then bring to a final volume of 2
L with ddH
2
O and mix. Label this solution “1x BT”.
28
Tris-buffered saline (TBS)
(20 mM Tris, 500 mM NaCl, pH 7.5)
Add 120 ml of 10x TBS to 1,080 ml of ddH
2
O. Label this
bottle “1x TBS”.
Wash solution (TTBS)
(20 mM Tris, 500 mM NaCl, 0.2% Tween 20, pH 7.5)
Add 2 ml of Tween 20 to 1 L of TBS. Label this solution
“TTBS”.
Avidin-AP or Streptavidin-AP solution
Add 100 µl of avidin-AP or streptavidin-AP to 100 ml TTBS.
Substrate solution for nitrocellulose membrane blots
Add 150 µl of the enhancer reagent to 3 ml of Immun-Star
chemiluminescent substrate. Label this solution “Substrate
Solution”.
For PVDF membrane blots, use the Immun-Star
chemiluminescent substrate without the enhancer reagent.
Total Protein Detection Procedure
Note: Before beginning, read through the entire procedure.
The following procedure is based on 100 ml of each solution,
which is sufficient volume to assay one 10 x 15 cm
membrane.
29
1. Wash the membrane in 100 ml 1x BT solution for 10 min.
If the membrane has been in a buffer containing amines,
repeat the wash two more times.
2. Decant the wash solution and replace it with fresh 1x BT
solution. While agitating the incubation vessel, add 200 µl
of NHS-biotin. Incubate the membrane for 15 min with
constant agitation. Do not prepare the BT solution
containing NHS-biotin before use, as the biotin
reagent is hydrolyzed in aqueous solution.
Note: To prevent hygroscopic accumulation of water in
the NHS-biotin reagent, equilibrate the vial to room
temperature before use and remove reagent with a sterile
syringe.
3. Wash the membrane in 100 ml of 1x BT for 5 min. Repeat
the wash step.
4. Wash the membrane in 100 ml of TTBS for 5 min. Repeat
the wash step.
5. Prepare the avidin-AP or streptavidin-AP solution.
Incubate the membrane in the solution for 1 hr with
agitation.
6. Wash the membrane in 100 ml of TTBS for 5 min. Repeat
the wash step.
7. Wash the membrane in 100 ml of TBS for 5 min. Repeat
the wash step.
30
8. Blot development – remove the membrane and drain
excess liquid without letting the blot dry. Place on a piece
of Saran wrap on a flat, level surface. Pipet the substrate
solution onto the membrane. Incubate 5 min. Remove the
membrane, draining the excess liquid from the blot. Seal in
a heat-sealable bag or in Saran wrap. Do not allow the
membrane to dry because this will result in a loss of signal.
9. Film exposure – expose X-ray or instant film to the blot
at RT. Exposure time will depend on the type of
membrane used and on the protein concentration.
Optimal conditions should be determined for each
particular application. Develop the film according to the
manufacturer’s instructions.
For further information, including a complete troubleshooting guide, consult the Biotin-Blot total protein detection kit
manual.
31
4006074 Rev D
Bio-Rad
Laboratories, Inc.
Life Science
Group
Web site www.bio-rad.com USA (800) 4BIORAD Australia 02 9914 2800
Austria (01)-877 89 01 Belgium 09-385 55 11 Brazil 55 21 2527 3454
Canada (905) 712-2771 China (86 21) 6426 0808
Czech Republic + 420 2 41 43 05 32 Denmark 44 52 10 00
Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 318 84-0
Greece 30 210 777 4396 Hong Kong (852) 2789 3300
Hungary 36 1 455 8800 India (91-124)-2398112/3/4, 5018111, 6450092/93
Israel 03 951 4127 Italy 39 02 216091 Japan 03-5811-6270
Korea 82-2-3473-4460 Latin America 305-894-5950
Mexico 55-52-00-05-20 The Netherlands 0318-540666
New Zealand 64 9 415 2280 Norway 23 38 41 30
Poland + 48 22 331 99 99 Portugal 351-21-472-7700
Russia 7 095 721 1404 Singapore 65-64153188
South Africa 00 27 11 4428508 Spain 34 91 590 52 00
Sweden 08 555 12700 Switzerland 061 717 95 55
Taiwan (886 2) 2578 7189/2578 7241 United Kingdom 020 8328 2000
Loading...