Bio-Rad Immun-Blot AP Colorimetric Kits User Manual

Immun-Blot

Assay Kit

Instruction

Manual

®

Table of Contents

Section 1 Preparation .................................................................... 1

1.1 Introduction ................................................................................ 1

1.2 Product Information.................................................................... 2

1.3 Materials Provided with the Immun-Blot Kit; Storage

and Stability of Kit Components ................................................ 4

1.4 Safety Instructions...................................................................... 5

1.5 Solutions..................................................................................... 6

Section 2 Immun-Blot Assay......................................................... 9

2.1 Experimental Strategy and General Recommendations............. 9

2.2 Detailed Assay Procedure........................................................... 11

2.3 Detailed Color Development Procedure..................................... 13

Section 3 Troubleshooting............................................................. 16

3.1 Tests for Monitoring Reagent Activity....................................... 16

3.2 Troubleshooting Guide............................................................... 17

Section 4 References ...................................................................... 21

Section 1
Preparation

1.1 Introduction

The Immun-Blot assay kits are enzyme immunoassay kits
optimized for the detection of specific antigens, at picogram levels,
following electrophoretic blotting or dot blotting to a membrane. The
kits provide all necessary components and chemicals, in an easy-to-use
form, for detection of selected purified proteins using a double
antibody or antibody-binding protein blotting assay.

The Immun-Blot alkaline phosphatase and Immun-Blot horseradish
peroxidase assay systems can be used to detect rabbit, mouse, or human
immune complexes.
antigens or cloned translation products following dot-blotting,
electrophoretic blotting,
or plaque lifts.

The Immun-Blot assay is fast and simple. Antigen is transferred
and bound to the membrane. This transfer can be done electro­phoretically, following separation of the antigen in a polyacrylamide or
agarose gel, or passively by either directly spotting the antigen to a
membrane or by overlaying a phage plate or lysed bacterial colony with
nitrocellulose membrane to bind expressed proteins. Following binding
of antigen, the remaining protein binding sites on the membrane
surface are blocked with gelatin or equivalent proteins which will not
react with the primary and secondary antibodies. BSA (not included in
the kit) is used as a blocker when the Bio-Dot
apparatus is used for blotting, since gelatin will obstruct filtration
through blotting membranes.

The membrane with bound antigen is then incubated with first
antibody, specific for the antigen to be detected. The membrane is

1

They will give sensible detection of specific

3-11

filter affinity transfers, 12and in situ colony

®

or Bio-Dot SF

2

1

washed to remove unbound antibody and incubated with the respective
GAR, GAM, or GAH second antibody, or Protein A or Protein G,
which has been conjugated to alkaline phosphatase (AP) or horseradish
peroxidase (HRP). In the case of AP, a color development reagent
containing 5-bromo-4-chloro-3-indoyl phosphate (BCIP) and nitroblue
tetrazolium (NBT) yields purple bands or “dots” on a white
background. The HRP color development reagent, containing 4-chloro­1-naphthol and hydrogen peroxidase, also yields purple bands against a
white background.

1.2 Product Information

Catalog
Number Product Description

Immun-Blot Assay Kit

170-6460 Goat Anti-Rabbit IgG (H+L) AP
170-6461 Goat Anti-Mouse IgG (H+L) AP
170-6462 Goat Anti-Human IgG (H+L) AP
170-6463 Goat Anti-Rabbit IgG (H+L) HRP
170-6464 Goat Anti-Mouse IgG (H+L) HRP
170-6465 Goat Anti-Human IgG (H+L) HRP
170-6466 Protein A-HRP
170-6467 Protein G-HRP

Individual Blotting Grade Reagents

170-6518 Goat Anti-Rabbit IgG (H+L) AP Conjugate
170-6520 Goat Anti-Mouse IgG (H+L) AP Conjugate
170-6521 Goat Anti-Human IgG (H+L) AP Conjugate
170-6515 Goat Anti-Rabbit IgG (H+L) HRP Conjugate
170-6516 Goat Anti-Mouse IgG (H+L) HRP Conjugate
172-1050 Goat Anti-Human IgG (H+L) HRP Conjugate
170-6522 Protein A-HRP Conjugate

Catalog
Number Product Description

Individual Blotting Grade Reagents (continued)

170-6425 Protein G-HRP Conjugate
170-6435 Premixed Tris Buffered Saline, 10x, 1 L
170-6431 Horseradish Peroxidase Conjugate Substrate Kit
170-6432 Alkaline Phosphate Conjugate Substrate Kit
170-6537 Gelatin, Blotting Grade, 200 g
170-6531 Tween-20, Blotting Grade, 100 ml

Blotting Membranes
Nitrocellulose Membrane (0.45 µm)

162-0115 Roll, 33 cm x 3 m, 1
162-0145 Sheets, 7 x 8.4 cm, 10
162-0117 Sheets, 9 x 12 cm, 10
162-0114 Sheets, 15 x 9.2 cm, 10
162-0116 Sheets, 15 x 15 cm, 10
162-0113 Sheets, 20 x 20 cm, 5

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

Blotting Media
Supported Nitrocellulose Membrane (0.45 µm)

162-0190 Sheets, 7 x 8.4 cm, 10
162-0191 Sheets, 10 x 15 cm, 10
162-0192 Sheets, 15 x 15 cm, 10
162-0193 Sheets, 20 x 20 cm, 10
162-0194 Roll, 30 cm x 3 m, 1

2

3

Catalog
Number Product Description

Supported Nitrocellulose Membrane (0.2 µm)

162-0195 Sheets, 7 x 8.4 cm, 10
162-0196 Sheets, 15 x 15 cm, 10
162-0197 Roll, 30 cm x 3 m, 1

PVDF Membrane (0.2 µm)

162-0180 Sheets, 10 x 15 cm, 10
162-0181 Sheets, 15 x 15 cm, 10
162-0183 Sheets, 20 x 20 cm, 10
162-0184 Roll, 24 cm x 3.3 m, 1
162-0185 Sheets, 20 x 20 cm, 3
162-0186 Sheets, 7 x 8.4 cm, 10

1.3 Materials Provided with the Immun-Blot Kit
Storage and Stability of Kit Components

Quantity Shelf

Product Description Provided Storage Life

Components supplied in all kits

Tris buffered saline, 10x, pH 7.5 1 L 4 °C >1 yr
Gelatin, Blotting grade 50 g RT >1 yr
Tween-20, Blotting grade 5 ml RT >1 yr
Conjugate solution* 0.5 ml 4 °C 1 yr

Components supplied in alkaline phosphatase kits

AP color reagent A (contains NBT in 10 ml -20 °C 1 yr
aqueous dimethylformamide [DMF],
containing magnesium chloride)

AP color reagent B (contains 10 ml -20 °C 1 yr
BCIP in DMF)

AP color development buffer, 25x 40 ml 4 °C >1 yr

Product Description Provided Storage Life

Quantity Shelf

Components supplied in horseradish peroxidase kits

HRP color regent A (contains 4-chloro- 200 ml -20 °C 1 yr
1-naphthol in diethylene glycol)

HRP color reagents B (contains 10 ml 4 °C 1 yr
hydrogen peroxide)

HRP color development buffer, 10x 100 ml 4 °C >1 yr

Additional components

Citrate buffered saline (CBS), 3x, 100 g RT >1 yr
pH 5.5 (provided in the Protein G HRP
kit only)

* Each kit provides a different conjugate solution. These reagents are shipped

frozen, and can be stored at -20 °C until their initial use. Once thawed, store the
conjugate solution at 4 °C. Avoid repeated freeze-thaw cycles, which will cause
breakdown of the product.

Note: Each kit contains all reagents necessary to complete 200 x 5 ml
assays. An excess of conjugate has been supplied in each kit. BSA must
be substituted for gelatin when blocking the membrane in the Bio-Dot
or Bio-Dot SF microfiltration apparatus.

1.4 Safety Instructions

Read entire instruction manual before beginning the assay.

1. Wear gloves and protective clothing, such as a laboratory coat and
goggles, when preparing and working with the solutions in the
assay. DMF, diethylene glycol, 4-chloro-1-naphthol, and BCIP can
cause skin and eye irritation, and contact should be avoided. In case
of contact, immediately flush the skin or eyes with copious
amounts of water for at least 15 minutes, and remove contaminated
clothing.

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5

2. Work in well-ventilated areas. Avoid inhalation of vapors when
handling solutions containing DMF, diethylene glycol, 4-chloro-1­naphthol, and BCIP.

3. Do not mouth-pipet any solutions.

1.5 Solutions

The following solutions must be prepared for each kit. The working
solution volumes are based on 20 assays developing 0.75 x 9.2 cm
nitrocellulose strips. For each strip, the assay uses 5 ml of each working
solution per incubation step. It is advisable to use at least 0.5 ml of
solution per square centimeter (cm
membrane must be completely covered with solution in all wash and
incubation steps. Larger volumes can be used for convenience, however
all volumes should be increased proportionately to insure that all kit
reagents are consumed at the same rate.

Stock solutions

Tris buffered saline, (200 mM Tris, 5 M NaCl, pH 7.5)
10x (10x TBS)

Color development HRP color development buffer, 10x
buffer AP color development buffer, 25x

Color development Ready to use as provided in the kit.
reagents

Citrate buffered With the Protein G-HRP assay kit, add the contents of
saline (CBS) the citrate buffered saline to a 1 L bottle, add 900 ml dd

water, agitate to dissolve, and quesce to 1.0 L with dd
water. Label this solution “3x CBS.” Store at 4 °C.

2

) of membrane. For best results, the

Working solution (based on 20 assays of 5 ml each):

Tris buffered saline (20 mM Tris, 500 mM NaCl, pH 7.5)
(TBS) Add 100 ml of 10x TBS to a 1 L bottle and quesce to

1.0 L with dd water. Label this bottle “TBS.”

Citrate buffered (20 mM citrate, 500 mM NaCl, pH 5.5)
saline (CBS) For the Protein G-HRP assay, add 100 ml of 3x CBS to

200 ml dd water. Label this solution “CBS.”

HRP Color The HRP color development buffer solution is a 10x
development buffer concentrate. It should be diluted with filtered, deionized

water. Dilute to a 1x working solution by adding 1 part
HRP color development buffer concentrate to 9 parts
filtered, deionized water. For example, to make 100 ml
of 1x HRP color development buffer, mix 90 ml of
filtered, deionized water with 10 ml of 10x HRP color
development buffer concentrate. Mix well. Store excess
solution at 4 °C.

Since the HRP color development buffer is better stored
for longer periods as a concentrate, make as much 1x
solution as is practical and needed for the planned
experiments.

AP Color The AP color development buffer solution is a 25x
development buffer concentrate. It should be diluted with filtered, deionized

water. Dilute to a 1x working solution by adding 1 part
AP color development buffer concentrate to 24 parts
filtered, deionized water. For example, to make 100 ml
of 1x AP color development buffer, mix 96 ml of
filtered, deionized water with 4 ml of 25x AP color
development concentrate. Mix well. Store excess
solution at 4 °C.

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7

Since the AP color development buffer is better stored
for longer periods as a concentrate, make as much 1x
solution as is practical and needed for the planned
experiments.

Wash solution (20 mM Tris, 500 mM NaCl, 0.05% Tween-20, pH 7.5)
(TTBS) Add 350 µl of Tween-20 to 700 ml to 1x TBS. Label

this bottle “TTBS.”
For the Protein G-HRP assay, make 400 ml of TTBS

instead, using 200 µl of Tween-20. Also prepare 300 ml
of TCBS by adding 150 µl of Tween-20 to 300 ml of
CBS. Label this bottle “TCBS.”

Blocking solution (3% gelatin - TBS)

Add 3.0 g of gelatin to 100 ml of TBS and heat to 50 °C,
with stirring, until dissolved.

A microwave oven will quickly solubilize the gelatin,
but do not heat above 65 °C. Label this solution
“Blocking solution - 3% gelatin in TBS.”

Antibody Buffer (1% gelatin - TTBS)

Add 2.0 g of gelatin to 200 ml of TTBS and heat, with
stirring to 50 °C until dissolved. Again, you can use a
microwave oven. Label this solution “Antibody buffer ­1% gelatin in TTBS.” Store at 4 °C.

For the Protein G-HRP assay, prepare only 100 ml of
1% gelatin in TTBS and prepare 100 ml of 1% gelatin
in TCBS.

First antibody Dissolve the first antibody to the appropriate titer in
solution 100 ml of antibody buffer. Label this solution “First

antibody solution.”

8

Second antibody Prepare the second antibody solution by dissolving 33 µl
conjugate solution of the antibody conjugate in 100 ml of antibody buffer,
or Protein A-HRP if applicable.
conjugate solution

Protein G-HRP Dissolve 33 µ l of the conjugate solution in 100 ml of
conjugate solution 1% gelatin in TCBS, if applicable.

Note: If a bacteriostat is needed, 0.01% thimerosal should be used.
Sodium azide is potent inhibitor of horseradish peroxidase.

Section 2
Immun-Blot Assay

2.1 Experimental Strategy and General
Recommendations

1. Background - Three types of background are common to immune

blot detection.
a. High membrane coloration - High membrane backgrounds

usually result when the blocking period is too short, when
Tween-20 is absent from the appropriate buffers and washes, or
when excessive amount of conjugate are used.

b. Non-specific antibody binding - Usually evidenced by extra

banding or high coloration in the separate lanes. This
background is usually due to impure or cross-reactive
antibodies, incubations with excessive antibody concentrations,
or an absence of Tween-20 from the appropriate buffers and
washes.

c. Non-specific conjugate binding - This background is

evidenced by band development in the absence of first antibody.
It results when conjugate is used in excess, or when Tween-20
is absent from the appropriate buffers and washes.

9

2. Temperature - All steps are performed at room temperature (22-

25 °C) unless indicated otherwise in the instructions. If a lower
assay temperature is required, it is advisable to double the
incubation and wash times for each 10 °C decrease in temperature.
In low temperature incubations, it may be necessary to substitute
BSA for gelatin.

3. Wash Purity - Poor quality water can contain inhibitors of

enzymatic color development. Use only deionized, distilled water
to prepare all solutions.

4. First 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 antibody buffer is used for
detection of antigens on the membrane surface. For
chromatographically purified monospecific antibodies, a 1:500­1:10,000 dilution in antibody 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 the greatest dilution of antibody reagent still resulting in
a strong positive signal without membrane background or
nonspecific reactions.

5. Conjugates - The conjugates supplied by Bio-Rad should be used

in the concentrations indicated in Section 1.5. Using a conjugate at
higher concentrations may result in an overall increase in
background without any increase in detection sensitivity.

6. 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.

7. Addition of detergents - Tween-20 is essential in washing to

eliminate overall background and non-specific hydrophobic

reactions. At 0.05%, Tween-20 will not disrupt binding of primary
antibodies to antigens or antigens to nitrocellulose, but will
optimize detection sensitivity by eliminating non-specific reactions.
Alternative detergents, or concentrations of Tween-20 other than

0.05% should not be substituted. The wash between blocking the
nitrocellulose with TBS - 3% gelatin and probing with first
antibody is essential and should not be altered.

8. Color development reagents - Immun-Blot assay kits are provided

with one of two color development reagent systems. All color
development reagents are ready to use after preparing a stock
solution of AP or HRP color development buffer.

9. Technical Service - Contact Bio-Rad Laboratories Technical

Services Group in Hercules, California, toll free 1-800-4BIORAD,
or your local Bio-Rad representative, if you require assistance.

2.2 Detailed Assay Procedure

Note: Before beginning, read through the entire procedure.

1. Antigen applications - 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 develop color if the
procedure is successful. This is an excellent check on the operation
of the assay and will help you to gauge the rate of color
development.

a. Dot-blotting - Cut the nitrocellulose sheet to appropriate size

(i.e. 0.9 x 9.2 cm strips, 3 x 5 cm rectangles, or Petri dish
circles). It is advisable to draw a grid on the membrane with a
pencil. Typical grids consist of 1 x 1 cm squares. Next, the dry
nitrocellulose is wetted by slowly sliding the membrane at a 45°
angle into TBS. Remove the thoroughly wetted membrane from
the TBS and dry it on filter paper for approximately 5 minutes.

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11

Apply sample antigen to each grid square using a syringe or a
variable pipette, by displacing 1 µ l of sample to the tip of the
syringe or pipette as a drop and gently touching it to the surface
of the nitrocellulose membrane. If the antigen sample is very
dilute, it is possible to apply successive 1 µ l doses at the same
spot by letting the previous sample application dry completely
before adding an additional dose. In all cases, the nitrocellulose
membrane should be allowed to dry completely before
proceeding to the blocking step.

b. Electrophoretic blotting - The anitgens of interest are

electrophoretically transferred to the membrane from a gel
support (i.e. SDS-PAGE gel, IEF gels, or native gels) using the
Trans-Blot

®

, Mini Trans-Blot, or Trans-Blot SD cell. If desired,
cut the wet nitrocellulose membrane into 0.6-0.8 cm wide strips.
Immerse the strips or the entire sheet in TBS proceeding to the
blocking step.

c. Microfiltration blotting - The Immun-Blot assay kits can easily

be adapted for use in the Bio-Dot or Bio-Dot SF apparatus.
These instruments allow rapid, reproducible applications of up to
96 samples on one membrane sheet. All applications and
washes, except color development, are carried out in the
apparatus.

2. Blocking step - After the antigen is applied, using one of the above

methods, immerse the membrane, at a 45° angle, into the blocking
solution. Gently agitate the solution using an orbital shaker platform
and incubate for 30 minutes to 1 hour at room temperature (RT).

3. Wash - Decant the blocking solution and add TTBS to the

membrane. Wash for 5-10 minutes with gently agitation at RT.

4. First antibody - Decent the TTBS and add first antibody solution

to the membrane. Incubate 1 to 2 hours with gently agitation.

Overnight incubation may be preferred, since longer incubation
periods may increase the sensitivity of detection.

5. Washes - Remove the unbound first antibody by washing the

membrane in TTBS for 5 minutes at RT. Decant the wash solution
and repeat the wash with another portion of TTBS for 5 minutes at
RT. Protein G-HRP assays, these washes should be done in TCBS
instead of TTBS. The optimal binding pH of Protein G is 5.5.
Therefore, the Protein G-HRP binding step should be carried out in
the citrate buffer.

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6. Conjugate binding step - Decant the TTBS and add the second

antibody solution, Protein A-HRP conjugate solution, or Protein G­HRP conjugate solution, and incubate 30 minutes to 2 hours using
gently agitation at RT. The optimum conditions of dilution and
incubation time must be determined experimentally.

7. Final washes - Remove the conjugate solution

*

, and wash the
membrane in TTBS for 5 minutes with gently agitation at RT.
Decant the solution and wash with another portion of TTBS for 5
minutes at RT. Just prior to color development, wash the membrane
in TBS for 5 minutes at RT with gentle agitation to remove residual
Tween-20 from the membrane surface.

* Save used conjugate solution for testing in case results are unsatisfactory. See

Section 3.1.

2.3 Detailed Color Development Procedure

Procedure for Alkaline Phosphatase Blots

1. Developer preparation - Immediately before use, add 1.0 ml of

AP color reagent A and 1.0 ml of AP color reagent B to 100 ml 1x
of AP color development buffer at RT. This solution can be stored
at 4 °C overnight, but prompt use is recommended.

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2. Color development - Immerse the membrane in the color develop-

ment solution.

**

Protein concentrations greater than 100 ng will
immediately become visible as purple bands or dots. Lower
concentrations of protein will take longer, but should be visible
within 30 minutes. To maximize sensitivity, the incubation in color
development solution can be extended to 4 hours. Should a large
amount of precipitate form before color development is complete,
decant the color development solution and add additional, freshly
prepared, color development solution. The precipitate, which is
usually generated by high concentrations of alkaline phosphatase
on the membrane surface, will settle on the membrane and can
produce unusually high backgrounds.

3. Washing - Stop the development by immersing the membrane in

dd water for 10 minutes with gentle agitation. Change the water at
least once during the 10 minute period to remove residual color
development solution.

4. Reading, drying, and storage - Take photographs while the

membrane is wet to enhance the purple color. Acceptable
photographs can be produced using Polaroid Type 108, Polacolor 2
Land Film, at f8 and 1 second exposure. Film should be developed
for 1 minute. Dry the membrane on filter paper and store between
polyester sheets. Protect from light to minimize fading.

** Save used color development solution for testing in case results are

unsatisfactory. See Section 3.1.

Procedure for Horseradish Peroxidase Blots

1. Developer preparation - Add 600 µl HRP color reagent B to 100

ml of 1x HRP color development buffer at RT. Add this solution to
20 ml of HRP color reagent A immediately prior to use. This
solution cannot be successfully stored. HRP color development
solution will precipitate out of solution if kept at 0-4 °C, and will

be inactivated with exposure to direct light. Warm the HRP color
development buffer to RT prior to addition of the color reagent
solutions. HRP color reagents A and B should be kept at their
proper storage temperature prior to mixing to maximize their
activity and prolong their shelf life. Protect the color development
solution from light.

2. Color development - Immerse the membrane in the color develop-

ment solution.* Protein concentrations greater that 100 ng will
immediately become visible as purple bands or dots. Lower
concentrations of protein will take longer, but should be visible
within 30 minutes. Avoid development periods longer than 45
minutes as bleaching or fading of color in positive reactions will
occur. If a heavy precipitate forms in the color development
solution, a fresh solution should be prepared and used immediately.

3. Washing - Stop the development by immersing the membrane in

dd water for 10 minutes with gentle agitation. Change the water at
least once during the 10 minute period to remove residual color
development solution.

4. Reading, drying and storage - Take photographs while the

membrane is wet to enhance the purple color. Acceptable
photographs can be produced using Polaroid Type 108, Polacolor 2
Land Film, at f8 and 1 second exposure. Film should be developed
for 1 minute. Dry the membrane on filter paper and store between
polyester sheets. Protect from light to minimize fading.

* Save used color development solution for testing in case results are

unsatisfactory. See Section 3.1.

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Section 3
Troubleshooting

3.1 Tests for Monitoring Reagent Activity

1. Activity test for the color development solution.

Combine 1.0 ml of the color development solution with 10 µ l of
full strength second antibody conjugate or Protein A or Protein G
conjugate. The color reaction should develop immediately. If color
fails to develop within a few minutes, the color development
solution is inactive. Make up fresh working solution and repeat the
color development assay.

2. Enzyme activity test for the conjugate solution.

Combine 1.0 ml of the color development solution (tested in step 1)
and 1.0 ml of the 1:3,000 dilution conjugate solution. A light blue
tinge should develop within 15 minutes. If color fails to develop
within 25 minutes, the conjugate solution is suspect. Repeat the
procedure with a freshly prepared dilution of conjugate.

3. Activity test for the first antibody solution.

Use a RID, Ouchterlony immunodiffusion, precipitation, or ELISA
test to determine reactivity of the antibody with the antigen. If
possible, repeat the Immun-Blot procedure with a more
concentrated first antibody solution.

3.2 Troubleshooting Guide

Problem Probable cause Recommended solution

1. No reaction or Color develop- Color development reagents
weak color ment solution must be stored at the proper
development is inactive (see temperature (see Section 1.3).

Section 3.1).

HRP color development solution
will precipitate out of solution if
kept at 0-4 °C, and detection
sensitivities may be decreased.
Warm the HRP color development
buffer to RT prior to addition of the
color reagent solutions. HRP color
reagents A and B should be kept at
their proper storage temperature
prior to mixing to maximize their
activity and prolong their shelf life.

Avoid bacterial contamination of
the color development buffers by
storage at 4 °C.

Tap water can inactivate the color
development solution. Use only
distilled, deionized water to
prepare the solutions.

First antibody Antibody is improperly stored.
solution is in- Avoid bacterial contamination,
active or non- heat inactivation, and repeated
saturating (see freeze-thaw cycles.
Section 3.1).

Antibody titer is too low. Increase
the concentration of antibody used
in the assay.

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17

Problem Probable cause Recommended solution

Tween-20 may affect the
reactivity of some antibodies.
Eliminate Tween-20 from the
assay (except the wash and after
blocking).

Conjugate is Conjugate is improperly stored.
inactive (see Store at 4 °C. Avoid heat inac­Section 3.1). tivation. Do not sub-ject to

repeated freeze-thaw cycles.
The concentration of conjugate is

non-saturating. Increase the
conjugate concentration used in
the assay.

Conjugate may be contaminated,
causing inactivation of the
antibody (of Protein A or Protein
G) or the enzyme.

Tap water may cause inactivation.
Use only distilled, deionized
water.

Azide is a potent inhibitor of
horseradish peroxidase. Use
thimerosal as a bacteriostat.

Little or no Tween-20 may wash bound
antigen is antigen from the membrane.
bound to the Eliminate Tween-20 from the
membrane. assay (except the wash after

blocking).

Problem Probable cause Recommended solution

Transfer of protein onto the
membrane was incomplete. Stain
the gel to assure transfer of
protein. Use prestained standards
to monitor transfer efficiency.
Consult the Trans-Blot, Trans-Blot
SD, or Mini Trans-Blot manual for
proper electrophoretic transfer
procedures. Refer to the Bio-Dot
or Bio-Dot SF manual for proper
dot blotting procedures.

First antibody Loss of activity may have oc­is not specific curred during electrophoretic
or does not rec- transfer. Pre-test the reactivi­ognize denat- ty of the antibody against
ured antigens both native and denatured
(common with antigen by a dot blot. Refer
monoclonals). to the Trans-Blot manual for the

transfer of native antigens.

Antigen is too Increase the amount of anti­dilute. gen in the assay.

2. High back- Blocking is Increase the blocking step to

ground (refer insufficient. 60 minutes.
to experimen­tal strategy Gelatin block- Prepare and use fresh block-
section) ing solution is ing solution.

old.
Nitrocellulose Remove the membrane when

is left in the the reaction appears to be
color develop- complete. If precipitate in
ment solution the color development
too long. solution appears, decant the

solution and use fresh reagent.

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19

Problem Probable cause Recommended solution

Blot is washed Tween-20 is necessary in the
in the absence washes to reduce back
of Tween-20. ground.

Conjugate is Use the recommended
used at an ex- 1:3,000 dilution.
cessive con­centration.

Contamination Refer to the Trans-Blot,
occurred dur- Trans-Blot SD, or Mini
ing transfer. Trans-Blot manual.

Use of poor Use pure nitrocellulose from
quality, mixed Bio-Rad.
ester nitrocel­lulose will cause
increased back­ground.

Section 4
References

1. Blake, M. S., Johnston, K. H., Russell-Jones, G. J. and Gotschlich, E. C.,

Anal. Biochem., 136, 175 (1984).

2. Hawkes, R., Niday, E. and Gordon, J., Anal. Biochem., 119, 142 (1982).

3. Towbin, H., Staehlin, T. and Gordon, J., PNAS, 76, 4350 (1979).

4. Glass, W. F., Briggs, R. C. and Hnilica, L. S., Science, 211, 70 (1981).

5. Glass, W. F., Briggs, R. C. and Hnilica, L. S., Anal. Biochem., 115, 219

(1981).

6. Karcher, D., Lowenthal, A., Thormar, H. and Noppe, M., J. Immunol.

Meth., 43, 175 (1981).

7. Reiser, J. and Wardale, J., Eur. J. Biochem., 114, 569 (1981).

8. Olmstead, J. B., J. Biol. Chem., 256, 11955 (1981).

9. Symington, J. M., Green, J. and Brackmann, K., PNAS, 78, 177 (1981).

10. Burnette, W. N., Anal. Biochem., 112, 195 (1981).

11. Legocki, R. P. and Verma, D. S. P., Anal. Biochem., 111, 385 (1981).

12. Erlich, H. A., Levinson, J. R., Cohen, S. N. and McDevitt, M. O., J. Biol.

Chem., 254, 12240 (1979).

13. Akerstrom, B. and Bjorck, L., J. Biol. Chem., 261, 10240 (1986).

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Bio-Rad
Laboratories

U.S. (800) 4BIORAD California [phone] Australia 02-9914-2800 Austria
(1) 877 89 01 Belgium 09-385 55 11 Canada [phone] China [phone]
Denmark 39 17 9947 Finland 90 804 2200 France (1) 49 60 68 34 Germany 089
31884-0 India 91-11-461-0103 Italy 02-21609 1 Japan 03-5811-6270 Hong Kong
7893300 The Netherlands 0318-540666 New Zealand 09-443 3099 Singapore (65)
272-9877 Spain (91) 661 70 85 Sweden 46 (0) 735 83 00 Switzerland 01-809 55 55
United Kingdom 0800 181134

Life Science
Group

SIG 020996 Printed in USA

LIT171 Rev D