CST Guide:

14
Section II: ANTIBODY APPLICATIONS
Western Blotting (WB)
Protein Detection
Western blotting (WB) or immunoblotting is the most common technique used for monitoring protein
expression in cells or tissue. WB can be used to measure total or modification-specific protein levels
qualitatively or quantitatively by chemiluminescent or fluorescent detection. The WB protocol comprises
multiple steps beginning with preparation of cell lysate and separation of cellular proteins by molecular
weight on a polyacrylamide gel matrix (SDS-PAGE). The separated proteins are then transferred to a
nitrocellulose or polyvinylidene fluoride (PVDF) membrane and detected using target-specific antibodies.
Prior to detection, the membrane is incubated in a blocking solution to prevent nonspecific antibody
binding. A wide variety of primary antibodies can be used to probe western blots including modificationspecific,
motif-specific, or total protein antibodies. Proteins can be detected indirectly using an
unconjugated primary antibody specific to the target of interest coupled with a secondary antibody
conjugated to either an enzyme (horseradish peroxidase or HRP) for chemiluminescent detection or an
infrared dye for fluorescent detection. Alternatively, primary antibodies directly conjugated to HRP can
be used to detect proteins in a one-step protocol.
The most common challenges presented by WB analysis are weak signal and high background.
The tips below will help you address the most common factors responsible for these problems.
WB Tips for Success
Incubate with primary antibody overnight
to improve antibody-target binding.
Overnight incubation of membrane and primary antibody can greatly enhance signal simply by allowing
more time for antibody-antigen binding. We have found that overnight incubation, with gentle agitation,
at 4°C results in stronger antibody specific signal.
Primary antibody incubation overnight at
4°C yields significantly increased antibody
binding compared to a 2 hr incubation.
Phospho-Akt (Ser473) Antibody #9271: WB analysis of extracts from HeLa
cells, untreated or treated with LY294002 #9901 or Human Insulin-like Growth
Factor I (hIGF-I) #8917, using #9271 (top) or PKC Antibody #2058 (bottom).
kDa
140
100
80
60
50
40
140
100
80
60
50
40
16 hr, 4°C
2 hr, RT
Phospho-
Akt (Ser473)
PKCδ
- - +
- - + LY294002
- + + - + + hIGF-I
Dilute secondary antibody in stronger
blocking agents to reduce nonspecific signal.
We suggest diluting secondary antibody in 5% nonfat milk in TBST rather than in 5% BSA in TBST. Milk
offers stronger blocking of nonspecific binding, hence the BSA-based dilution yields significantly higher
background than the milk-based dilution.
Diluting secondary antibody in milk yields
lower background levels because milk is a
stronger blocking agent.
Phospho-Stat3 (Tyr705) Antibody #9131: WB analysis of extracts from Jurkat
cells, untreated or treated with Human Interferon-α1 (hIFN-α1) #8927, using #9131.
Blots were incubated in Anti-rabbit IgG, HRP-linked Antibody #7074 diluted in either
5% nonfat milk in TBST or 5% BSA in TBST, as indicated.
kDa
200
140
100
80
60
50
40
Milk
BSA
– + – +
Weak signal of mid-to-high molecular weight proteins.
chapter 14: Western Blotting (WB)
Phospho-
Stat3
Due to their large size, high molecular weight proteins do not typically transfer as efficiently as smaller
proteins. Wet transfer often provides better transfer of mid-to-high molecular weight proteins than
semi-dry or dry transfer methods. Additionally, because methanol acts as a fixative, decreasing the
methanol concentration in transfer buffer from 20% to 5% and increasing transfer time to 3 hours can
also improve transfer efficiency of proteins >200 kDa.
Wet transfer provides better capture of mid-to-high
molecular weight proteins and a stronger WB signal.
Phospho-Stat3 (Tyr705) (D3A7)
XP ® Rabbit mAb #9145: WB
analysis of extracts from HeLa
cells, untreated or treated with
Human Interferon-α1 (hIFN-α1)
#8927, using #9145 or a
phospho-Stat3 (Tyr705) antibody
from a competitor. Blots were
transferred using either traditional
wet transfer methods (left) or
iBlot ® dry transfer system (right).
kDa
200
140
100
80
60
50
40
30
HeLa
Wet Transfer
Dry Transfer
HeLa HeLa HeLa
hIFN-α1
Phospho-
Stat3 (Tyr705)
Sonicate lysates to increase nuclear protein recovery.
20
kDa
100
80
Sonication thoroughly disrupts both cell and nuclear membranes, improving cell lysis and resulting in
higher protein levels in clarified lysate. This enhanced membrane disruption is especially important
for nuclear proteins, which may not be completely released through detergent use alone. If you do not
have access to a probe sonicator, passing samples through a fine gauge needle will also serve to break
membranes and shear DNA.
– +
CST #9145
– + – + – +
Alternate
Provider
CST #9145
Alternate
Provider
hIFN-α1
60
50
40
30
Lysate sonication is critical to the observation
of nuclear and chromatin-bound proteins.
20
10
-
Sonicated
+ - + sorbitol
Unsonicated
Phospho-
Histone H3
(Ser10)
Phospho-Histone H3 (Ser10) Antibody #9701: WB analysis of extracts from CKR/PAEC cells,
untreated or treated with sorbitol and either sonicated or without sonication, using #9701.
WB Protocols and Troubleshooting Videos
For more in-depth help with WB, please see our online WB Protocols and Troubleshooting videos.
www.cellsignal.com/wbvideo
226 For Research Use Only. Not For Use in Diagnostic Procedures. See pages 302 & 303 for Pathway Diagrams, Application, and Reactivity keys.
www.cellsignal.com/cstwb
227