Can I use SuperSignal West Femto Substrate with the identical protocol (i.e., same dilutions) I used with the SuperSignal West Pico Substrate?
No, all three SuperSignal Substrates require different dilutions. If you follow the SuperSignal West Pico Substrate protocol when you use SuperSignal West Femto Substrate, you will see high background or a decreased signal intensity and or duration. You must use less (more dilute) primary and secondary antibodies with SuperSignal Femto Substrate (see product instructions for recommended ranges).
How much more sensitive is SuperSignal West Femto Substrate than the other enhanced chemiluminescent (ECL) substrates?
SuperSignal West Femto Substrate is about 25- to 50-fold more sensitive than GE Healthcare ECL Substrate or Pierce ECL Substrate (Product # 32106). SuperSignal Femto Substrate is also significantly more sensitive than available chemiluminescent substrates for alkaline phosphatase.
Is milk a good blocking reagent to use with SuperSignal West Femto Substrate?
Yes and no. Milk contains variable amounts of biotin so it should not be used with avidin/biotin detection systems. Milk also contains varying amounts of phosphoproteins that may make interfere with anti-phosphotyrosine procedures. A variety of blocking buffers are compatible with the substrate.
What is the best blocking reagent?
Background is a relative phenomenon - no blocker will prevent all interactions 100% of the time. While a particular blocking agent may give a perfect signal-to-noise ratio for one set of reaction conditions, it may not work as well for another set of similar conditions. The key is to optimize the system by trying various blocking conditions.
Should any reagents be avoided when using of SuperSignal West Femto Substrate?
Yes. Avoid using sodium azide during and after probing steps involving horseradish peroxidase (HRP), as this will inhibit HRP activity. Sulfide, cyanide, fluoride and superoxide ions also inhibit HRP to some extent.
What is the lower detection limit?
The lower detection limit of SuperSignal West Femto Substrate is low-femtogram (1 x 10^-15).
Can the blots detected with this substrate be reprobed?
Although blots detected with chemiluminescent substrates can be stripped and reprobed, some antigen/antibody systems are sensitive to the stripping procedure and might not yield the same quality of results on a stripped blot compared to a new blot. Only actual experimentation will yield information as to whether a given system will allow reprobing.
What protocol should I use to strip and reprobe a blot?
See Tech Tip # 23: Strip and reprobe Western blots.
Why do I see more bands than I've seen in the past with other substrates?
Because this substrate is more sensitive than other chemiluminescent substrates you might detect low-abundance proteins that were not visible before. When using a more sensitive substrate, more careful optimization of blocking buffer steps and antibody concentrations is required.
Why is the entire film black or showing a reversed image?
The antibody concentration (primary and secondary) is too high. Use the antibody dilutions described in the product instructions. Most background will disappear when the proper blocking reagent and a HRP conjugate concentration are used.
Will SuperSignal West Femto Substrate work with nucleic acid blots (Southern blotting, etc.)?
Yes. However, SuperSignal West Femto Substrate was optimized for use in Western blots. For Southern and Northern blotting, use our North2South Chemiluminescent Nucleic Acid Hybridization and Detection Kit (Product # 17097).
With what types of membrane work best with SuperSignal West Femto Substrate?
Most researchers use nitrocellulose or polyvinyldiflouride (PVDF) membranes with SuperSignal West Femto Substrate. Both work well, although nitrocellulose seems to be better suited in some applications than PVDF. In addition, charge-modified nylon membrane performs well with this substrate.