Skip to main content

CDB Equipment Resource

Odyssey CLx Infrared Scanner

Location: U3202 MRB3

Purchased in July 2015, the CDB Equipment Resource currently has an Odyssey CLx model with the latest version of ImageStudio. Please ask the Equipment Manager for more information to learn the instrument and get access to the software package.

The VBI Instrument Core has one additional Odyssey CLx in MRB3.

Below, you will find helpful PDFs for the frequent Odyssey users. All of the information comes directly from Li-Cor as they have a truly extensive (and easy to access) collection of resources available online.

Some quick tips from a Li-Cor Application Support Representative:

Membranes:

If using Nitrocellulose membranes, any brand may work just fine.
However, if you are using PVDF membranes, make sure that they are specific for fluorescence.
There is much more variability between vendors of PVDF membranes and have found if it is not specific for fluorescence, there will be a lot of background.
Li-Cor has performed a lot of testing, as seen in Good Westerns Gone Bad, and found Immobilon FL to be the best option for PVDF membranes.

Blocking Buffer:

  • Blocking can be done in the Odyssey Blocking Buffer, Casein, or 5% Milk
  • DO NOT USE BSA! It is known to yield a high background.
  • The membrane should not be exposed to Tween-20 until blocking is completed, as high background will result.  However, Tween can be added back in with the washes and should be included in the antibodies.

Primary Antibody:

  • Add 0.2% Tween to primary and secondary antibody dilutions.
  • Li-Cor keeps an internal database of conditions from peer-reviewed publications on some targets

Secondary Antibody:

  • Dilute secondary antibodies to start with at around 1:15,000 for IRDye 680RD and IRDye 800CW secondary, or 1:20,000 for IRDye 680LT secondary.
  • Higher concentrations may result in very high background. Incubation is for 1 hour at room temp.
  • For PVFD, SDS (final concentration of 0.01%) and Tween® 20 (final concentration of 0.2%) must be added during the detection incubation step to avoid non-specific background staining.
Odyssey CLx Manual
Odyssey Quick Start
Good Westerns Gone Bad: Tips to Make Your NIR Western Blot Great
Western Blot Normalization: Challenges and Considerations for Quantitative Analysis
Protein Electrotransfer Methods and the Odyssey® Imaging Systems
Odyssey Western Blot Blocker Optimization
Complete CLx Application Protocols Manual

Hints & Tips from another rep:

The following are some hints and tips developed by a Li-Cor technical representative. You may want to consider these when using near-IR Fluorescence for the first time:

  • It is extremely helpful to run a comparison experiment, with one blot in your traditional format (i.e. ECL) and the other with the IRDye protocol.
    • This will allow for side-by-side comparisons to your current method.
  • Two-color detection requires primary antibodies from different host species.
  • Do not write on your gels with pen. Please use pencil.
  • If using blue ladder (MW Marker), use very small amounts
    • (2-3 µL is sufficient to see on the Odyssey).
  • Do not add Tween to blocking buffer until after blocking.
  • Blocking can be performed using LI-COR Blocking Buffer (BB; recommended) or milk.
    • Milk works well with nitrocellulose, but not so well with PVDF.
    • Our BB may result in better sensitivity.
    • If you have time, please try a blot in each, milk and Odyssey BB.
  • Do not use BSA for blocking; it decreases sensitivity quite a lot.
  • Adding Tween to antibody dilutions is recommended.
    • Typically between 0.05% and 0.2% is adequate.
    • 0.2% is most common.

Adding SDS to secondary antibody dilutions is recommended to reduce background and non-specific binding (PVFD Only).

  • To blocking buffer, add Tween 20 to a final concentration of 0.2% and SDS to a final
    concentration of 0.01%.

    • Dilute antibodies in LI-COR BB (if used to block) or PBS/TBS.
    • Try both PBS and TBS as there can be a difference in their effective blocking ability.
    • Dilute and incubate primary antibodies as you typically do.
  • Dilute secondaries between 1:5,000 to 1:25,000.
    • To start, I typically recommend 1:20,000.
    • Based on the results, optimum dilution can be tweaked.
    • Incubation is for 1 hour at room temp.
  • Handle membranes carefully and with forceps.
    • This is very important when using the secondaries.
    • A rinse of the forceps in ethanol after use in secondary dilutions is recommended.
  • I recommend a dilution volume of up to 10 mL for the secondary.
  • The dyes are stable.
    • Membranes can be prepared and then stored either dry or wet at 4° C for scanning at a later time.
  • Drying membranes after secondary incubation, particularly nitrocellulose will yield higher sensitivity, but you will not be able to strip the membranes.
  • Be aware that the labeled antibodies are light sensitive.
    • Keep them covered when incubating (by covering the dish with foil or a box).
  • There are no special requirements or procedures for coomassie stained gels.