Immunodetection
ProtocolBulletin 6219

Immunodetection
1

Wash — following transfer or protein application, wash the membrane
for 5–10 min in TBS.

5–10 min2

Block — incubate the membrane for 1 hr in blocking solution.

1 hr

32×
5–10 min4

Wash — wash the membrane twice in TTBS, 5–10 min per wash.

Primary antibody — dilute the antibody in antibody dilution or blocking
solution (refer to the instructions for the antibody for the recommended
final concentration). Incubate the membrane for 1–2 hr in the primary
antibody solution with gentle agitation.

1–2 hr2–6×
5–10 min

5Wash — wash the membrane 2–6 times in TTBS, 5–10 min per wash.

1 hr

67

Antibody conjugate — dilute the conjugate in TTBS (refer to theinstructions for the conjugate for the recommended final concentration).
Incubate the membrane for 1 hr in the enzyme conjugate solution withgentle agitation.

Wash — wash the membrane 3–6 times in TTBS, 5–10 min per wash.

3–6×
5–10 min

8Final wash — wash the membrane in TBS to remove the Tween 20
from the membrane surface prior to blot development and imaging.

1×9Signal development — for colorimetric development, add detection
substrate and incubate for 5–30 min depending on the specificreagents used. For chemiluminescence/fluorescence, see next page.

5–30 min

10Image, dry, and store — image the blot on a CCD laser-based imager,
or expose to X-ray film or instant photographic film. Develop the film
according to the manufacturer’s instructions.

Notes for Multiplex Detection
Gel equilibration removes contaminating electrophoresis
buffer salts. If not removed, these salts increase theconductivity of the transfer buffer and the amount of heatgenerated during transfer.

Equilibration is not necessary (i) when the same buffer isused for both electrophoresis and transfer (for example,
native gel transfers), or (ii) when using rapid semi-dry transfer
systems such as the Trans-Blot® Turbo™ system (consult theuser manual for the system you are using).

Equilibration also allows the gel to adjust to its final size prior
to electrophoretic transfer. Gels shrink or swell to various
degrees in the transfer buffer depending on the acrylamide
percentage and the buffer composition.

© 2011 Bio-Rad Laboratories, Inc.

Bulletin 6219

Notes for Chemiluminescence Detection
Follow steps 1–8 of the immunodetection assay, except
use more stringent washes (steps 5 and 7). Wash themembrane six times for 10 min each at these steps,
with strong agitation and a large volume of buffer toreduce background. Then follow below for step 9:

AB

Place the membrane protein-side up on a clean
piece of plastic wrap or a plastic sheet protector.
Add chemiluminescent substrate solution. Use atleast 0.1 ml per cm2 of membrane (about 6 ml fora standard 7 × 8.5 cm gel).

Note for Protein G-HRP Detection
Follow steps 1–4 on previous page. For step 5 (wash), use
TCBS instead of TTBS and then continue with steps 6–10.
Notes for Amplified Opti-4CN™ Detection
Follow steps 1–8 of the immunological assay on
previous page. Then:A
B
C

Incubate the membrane in diluted BAR for 10 min.
Wash the membrane 2–4 times in 20%
DMSO/PBST for 5 min each time.
Wash 1–2 times in PBST for 5 min. each time.

CIncubate the membrane for 3–5 min in thechemiluminescent substrate solution.

DIncubate the membrane and dilutedstreptavidin-HRP for 30 min.

D

Drain excess liquid from the blot and seal themembrane in a bag or sheet protector.

E ash the membrane twice in PBST forW
5 min each time.

EImage the blot on a CCD imager such as a
ChemiDoc™ or VersaDoc™ system, or expose to
X-ray film (for example, Kodak XAR or BioMax)
or instant photographic film, such as Polaroid
Type 667 or 612. Typical exposure times are 30 sec
to 5 min. Develop the film according to themanufacturer’s instructions.

Notes for Fluorescence Detection
Follow steps 1–8 of the immunodetection assay. Imaging ofmost fluorescent dye conjugates (Cy, Dylight, Alexa Fluor,and IRDye dyes) can be performed on wet or dry membranes.
Imaging of fluorescent protein conjugates (phycoerythrin,allophycocyanin) should be performed on wet membranes
for maximum sensitivity. Refer to the table below forrecommended imager settings. Excitation and emission
wavelengths are similar for non-Bio-Rad imagers as well.								Red excitation(e.g., Alexa647, Cy5,DyLight 649)

Blue excitation(e.g., FITC,Alexa 488,DyLight 488)

Green excitation(e.g., Alexa 555,Cy3, DyLight 548,TAMRA)

VersaDoc MP
PharosFX™

695 BP
635 Ex/695 BP

530 BP
488 Ex/530 BP

605 BP
532 Ex/605 BP

FContinue with steps 9–10.

Notes for Amplified AP Detection
Follow steps 1–5 of the immunodetection assay on previouspage. Then:AB

Incubate the membrane for 1–2 hr in biotinylated
secondary antibody solution.
While the blot is incubating in the biotinylated
antibody solution, prepare the streptavidinbiotinylated AP complex. Allow the complex toform for 1 hr at room temperature.

C ash the membrane twice in TTBS, 5–10 min
Wper wash.

DIncubate the membrane for 1–2 hr in the streptavidin
complex solution.

EContinue with steps 7–10.
TIPS
If kept wet, blots using
HRP or AP conjugates canbe stored for several days
prior to development andimaging. Leave blot in TBS,or place membrane between
two pieces of filter paper
soaked in TBS, and place ina sealable container.

© 2011 Bio-Rad Laboratories, Inc.

Bulletin 6219

This is an excerpt from Bio-Rad’s comprehensive Protein Blotting Guide (Bulletin 2895).

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Life ScienceGroup

Bulletin 6219 Rev A

US/EG11-0864

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