 | Get the Best Sensitivity with ECL Advance
Introduction
ECL Advance™ Western Blotting Detection Kit was directly compared to Pierce SuperSignal West Femto Maximum Sensitivity Substrate using manufacturer’s recommended conditions.
The aim of the study was to show the difference in antibody concentrations required for both systems to exhibit the highest sensitivity level attainable within a given antibody system.
ECL Advance uses the same protocol as ECL™ and ECL Plus™ with the exception of minor changes to the antibody incubation steps and the use of a specifically developed and optimized blocking agent.
The high intensity of signal produced by ECL Advance enables antibodies to be used at lower concentrations, making experimental work much more cost effective. This is especially important where rare or difficult to produce antibodies are employed.
Products used
Amersham Biosciences products used for this process:
Hybond ECL RPN2020D
ECL Blocking Agent RPN2125
ECL Advance Western Blotting Detection Kit RPN2135
Anti-mouse IgG HRP NA931
Other materials required
· Actin from bovine muscle (Sigma A-3653)
· Anti-actin (Sigma, A-4700)
· Phosphate buffered saline (PBS), 10 mM
· Tween 20
Method
Actin (from bovine muscle) was loaded onto a pair of 12% polyacrylamide gels in doubling dilutions starting at 50 ng. Following electrophoresis (150 V for 45 min) the samples were transferred (at 30 V overnight) to two Hybond™ ECL membranes (nitrocellulose).
Following transfer, detection was performed with each of the respective detection kits (see protocols below). The blots were immuno-detected using the recommended protocols for each kit and imaged on high sensitivity chemiluminescent film (Hyperfilm™ ECL). The wash buffer and block/antibody diluent used was 0.1% Tween™ 20 (v/v) in 10 mM PBS (PBST).
ECL Advance Western Blotting Detection Kit Protocol
1. Block for 1 h in ECL Advance Blocking Agent.
2. Rinse twice in PBST.
3. Incubate for 1 h in anti-actin diluted 1:50 000 in ECL Advance Blocking Agent.
4. Rinse twice in PBST.
5. Wash for 15 min in PBST followed by two further 5 min washes.
6. Incubate for 1 h in anti-mouse IgG HRP diluted 1:200 000 in ECL Advance Blocking Agent.
7. Rinse twice in PBST.
8. Wash for 15 min in PBST followed by two further 5 min washes.
9. Mix equal amounts of the ECL Advance substrates and incubate for 5 min.
10.Drain the blots, wrap in SaranWrap, and expose to Hyperfilm ECL for 5 min.
SuperSignal West Femto Maximum Sensitivity Substrate Protocol
1. Block for 1 h in 5% milk block.
2. Rinse twice in PBST.
3. Incubate for 1 h in anti-actin diluted 1:5000 in PBST.
4. Rinse twice in PBST.
5. Wash for 15 min in PBST followed by two further 5 min washes.
6. Incubate for 1 h in anti-mouse IgG HRP diluted 1:100 000 in PBST.
7. Rinse twice in PBST.
8. Wash six times for 5 min each in PBST.
9. Mix equal amounts of the detection reagents and incubate for 5 min.
10.Drain the blots, wrap in SaranWrap, and expose to Hyperfilm ECL for 5 min.
ECL Advance Western Blotting Detection Kit | SuperSignal West Femto Maximum Sensitivity Substrate |
 |  |
Primary antibody (anti actin) 1:50 000
Secondary antibody (anti-mouse IgG HRP) 1:200 000
Membrane Hybond ECL
Hyperfilm ECL exposure: 5 min | Primary antibody (anti actin) 1:5000
Secondary antibody (anti-mouse IgG HRP) 1:100 000
Membrane Hybond ECL
Hyperfilm ECL exposure: 5 min |
Fig 1. Western blotting performance of ECL Advance Western Blotting Detection Kit and SuperSignal West Femto Maximum Sensitivity Substrate
Discussion
Optimization of ECL Advance and SuperSignal West Femto Maximum Sensitivity Substrate kits indicated that the use of stronger antibody concentrations produced a higher background noise level without detection of any lower quantity actin bands (data not shown). The images shown therefore represent optimal conditions for detection of actin with each of the two kits when using their respective recommended conditions.
Even when 10 times less primary antibody and half as much secondary antibody is used, ECL Advance provides higher sensitivity results than SuperSignal West Femto Maximum Sensitivity Substrate (Fig 1).
Conclusion
These results highlight the differences in antibody concentrations required to produce optimal results between the two kits. The results also exemplify the fact that ECL Advance is particularly suited to Western blotting applications where the primary antibody employed is in short supply or expensive to produce.
Legal
ECL, ECL Advance, ECL Plus, Hybond, and Hyperfilm are trademarks of Amersham Biosciences UK Limited. Amersham and Amersham Biosciences are Trademarks of GE healthcare. ECL Advance was developed by Lumigen Inc. and is exclusively distributed by Amersham for Western blotting under license from Lumigen. SuperSignal West Femto Maximum Sensitivity Substrate is a registered trademark of Pierce Biotechnology Inc. Rockford IL. Tween is a trademark of ICI Americas Inc.
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