Create a Work Area and Import an Image into this Free Western Blot Analysis Software

In this series of posts, you will discover how easy it is to use Image Studio™ Lite. This free Western blot analysis software from LI-COR® allows you to easily create your own work area and then import images from numerous sources. So, if you have an old film scan, or an image from another chemiluminescent Western blot imaging system, try using this free Western blot analysis software. In subsequent posts, we will talk about the other functionalities of Image Studio Lite.

The first video in this post will show you how to open Image Studio Lite software and create a work area. (If you need to find out the installation requirements for Image Studio Lite, see the 17-Jul-13 Post, Computer Requirements to Get Started with Free LI-COR® Image Studio Lite Software).

This second video walks through the steps needed to import an image into Image Studio Lite. Image Studio Lite analyzes images in the tif, png, or jpeg format as well as images acquired with past versions of the Odyssey® or Pearl® imaging systems software.

Download FREE Image Studio Lite Western Blot Analysis Software from LI-COR and get started right away! (Bonus points for telling your friends about it too! :-))

Image Studio Lite – A Dream Come True without the Unicorn!

Multiplex Western Blotting System Turbo-Charges Western Blot Results Output

Example of Multiplex Western Blotting using the MPX Blotting SystemMultiplex Western blotting is a powerful tool that allows you to get more out of your Western blots. Multiplex detection becomes possible when you utilize the MPX™ (Multiplex) Blotting System and LI-COR IRDye® near-infrared fluorescent dye-labeled secondary antibodies.

Multiplex Westerns can be imaged on any of the Odyssey® Imagers and provide results for a possible maximum of 48 targets on a single membrane — 24 per channel with two-color detection — and the option for quantitative analysis, saving you time and reagents! The MPX Blotting System can be used if you need to optimize:

  • Primary antibodies – to determine the primary antibody that has the right specificity and the right dilution for use
  • Antibody incubation times
  • Blocking conditions – which blocking buffer will give you the optimum results
  • Secondary antibodies – what dilutions is best to use without getting a lot of non-specific binding?
  • Or just about anything else you need to optimize!

Watch this 4 minute video on how easy it is to get the most out of multiplexing with the MPX Blotting System. You can also download the handy MPX Blotter User Guide.

5 Technical Tips for Chemiluminescent Western Blotting Success with the C-DiGit® Scanner

In this short video, Jessica talks about 5 tips to help ensure that imaging chemiluminescent Western blots on the C-DiGit Blot Scanner is a success – the first time and always!

Here’s a recap of the tips of those five technical tips:

  1. Install Image Studio™ Software on your computer before connecting the C-DiGit Blot Scanner.
  2. Incubate using room temperature substrate.
  3. Wrap your blot so it stays wet during the scan.
    1. Remember to place your blot protein side down.
    2. If sensitivity is an issue, use WesternSure® PREMIUM or SuperSignal® West Femto Chemiluminescent Substrate.
  4. Start with high sensitivity scan for your first scan and then work from there.
  5. Image on the C-DiGit Scanner first and then exposure your film.

C-DiGit Blot ScannerHappy Blotting!

WesternSure® Chemiluminescent Western Blotting Reagents from LI-COR®

WesternSure Chemiluminescent Western Blotting ReagentsDetect your Western Blots with Confidence! Use NEW! WesternSure® Chemiluminescent Reagents from LI-COR!

Now, in addition the great imaging systems for chemiluminescent Western blots, LI-COR Biosciences offers chemiluminescent Western blotting substrates and HRP-conjugated secondary antibodies for use in performing your BEST chemiluminescent Western blots ever. And, the WesternSure Pen is used to annotate visible protein ladders prior to chemiluminescent Western blot detection.

WesternSure chemiluminescent Western blotting reagents offer the best performance available when compared to other competitive products on the market. WesternSure PREMIUM Chemiluminescent Substrate is a highly sensitive enhanced substrate for detecting horseradish peroxidase (HRP) on immunoblots.

WesternSure HRP-conjugated secondary antibodies (Goat anti-Mouse and Goat anti-Rabbit) are compatible with a variety of chemiluminescent substrates and are optimized for use with WesternSure PREMIUM chemiluminescent substrates.

Happy Blotting!

NEW! IRDye® Goat Anti-Mouse IgM Secondary Antibodies from LI-COR®!

IRDye Dye-labeled Goat anti-Mouse AntibodiesOur IRDye secondary antibody line is growing! We have recently added IRDye Goat anti-Mouse IgM (μ chain specific) secondaries labeled with:

  • IRDye 800CW (PN 926-32280)
  • IRDye 680RD (PN 926-68180) or
  • IRDye 680LT (PN 926-68080).

Just like all of the LI-COR IRDye secondary antibodies, these are highly cross-adsorbed secondary antibody conjugates suitable for a variety of applications (see the table below).

IRDye 800CW secondary antibodies are the antibodies of choice for a wide variety of applications in the 800 nm channel (see the list below). IRDye 800CW secondary antibodies can be used for 2-color detection when multiplexed with IRDye 680RD or IRDye 680LT secondary antibodies.

IRDye 680RD secondary antibodies are the antibodies of choice for In-Cell Western Assay and Western blot applications in the 700 nm channel. These antibodies can be used for 2-color detection when multiplexed with IRDye 800CW secondary antibodies. These antibodies are our most universal use 700 nm channel antibodies. Start using IRDye 680RD first over other 700 nm dyes. Dilution working range 1:10,000 – 1:40,000.

IRDye 680LT secondary antibodies have been proven the brightest signal for Western blot detection in the 700 nm channel and are comparable to Alexa Fluor 680 secondary antibodies. Choose IRDye 680LT secondary antibodies to get high signal and for specific uses of detection in the 700nm channel. These antibodies are not recommended when getting up and running on system. Once established near-infrared protocols are optimized with IRDye 680RD, IRDye 680LT can be used to optimize signals in the 700 channel. Dilution range 1:20,000 – 1:40,000. Note: optimization may be required with IRDye 680LT.

Application IRDye 800CW
Secondaries
IRDye 680RD
Secondaries
IRDye 680LT
Secondaries
Western Blot
In-Cell Western™ Assay Not Recommended
On-Cell Western Assay Not Recommended
Protein Array
Immunohistochemistry
Microscopy
2D Gel Detection
Tissue Section Imaging
Small Animal Imaging Not Recommended
Virus Titration Assay Not Known Not Known
FRET-based Assay Not Known Not Known


Note: Now, as of December 15, 2014, you can also get 0.1 mg sizes of all of our IRDye dye-labeled secondary antibodies. Check out our complete listing here and our new filtering tool!

Reprobe Fluorescent Westerns with NewBlot™ Western Blot Stripping Buffers

NewBlot Stripping Buffer IconNewBlot Western Blot Stripping Buffers are specially formulated for use with IRDye® infrared dyes (680RD, 680LT, and 800CW only) and the Odyssey® Infrared Imaging Systems to help you save time and money on recreating samples. NewBlot Stripping Buffer allows you to reuse the same blot by stripping and reprobing up to two fluorescent antibodies simultaneously.

So, you may ask, what’s so great about NewBlot Stripping Buffer?

  • Effectively removes antibodies, yet gentle enough to retain immobilized proteins
  • Strips blots at room temperature in 20 minutes or less without an unpleasant odor
  • Allows you to reuse the same blot up to 3X! (see the data below!)
  • Offers qualitative analysis after stripping

In the example below, beta tubulin and ERK2 were run on a gel and transferred to Immobilon®-FL PVDF membrane. They were probed with primary antibodies rabbit anti-beta-tubulin and mouse anti-ERK2 and then with IRDye 680 Goat anti-Rabbit (red) and IRDye 800CW Goat anti-Mouse (green), respectively. NewBlot PVDF Stripping Buffer was used to strip the blot, which was then reprobed with the fluorescent secondary antibodies. This was repeated 2 more times. As you can see from the series of images, there is very little apparent loss of signal in either channel in the third blot as compared to the original blot.
Example of using NewBlot Stripping Buffer on PVDF Membranes

NewBlot is available in two ‘flavors’: one for stripping nitrocellulose membranes and the other for stripping PVDF membranes.

Get more power out of your blot with NewBlot Western Blot Stripping Buffers!

Note: On August 25, 2014, we launched two new Western blot stripping buffers: NewBlot™ IR Stripping Buffer for infrared Western blots on either PVDF OR nitrocellulose membranes; and, WesternSure® ECL Stripping Buffer for chemiluminescent Western blot stripping and reprobing. BOTH do not require hazardous shipping charges, unlike many other Western stripping buffers.

Which Western Blot Detection Method Should You Use?

Western blots can be detected with fluorescent, chemiluminescent, or colorimetric methods. Which Western blot detection method should you choose? Find out how the three common Western blot detection methods compare to each other in terms of time, sensitivity, and other important factors. The, choose what works best for your research.

Fluorescent detection: Fluorescent detection uses secondary antibodies labeled with fluorescent dyes, rather than enzymes. No substrates are needed.

Enzymatic detection: Chemiluminescent and colorimetric methods use secondary antibodies labeled with enzyme reporters such as horseradish peroxidase (HRP). Signal-generating substrates are used.

Fluorescent detection uses NIR fluorescent dyes to generate a signal.
Secondary antibodies are labeled with dyes such as IRDye 800CW or IRDye 680RD
• Digital imaging reveals target protein signals with high sensitivity
• Quantitative (signal is proportional to the amount of target protein present)
• Stable fluorescent signals are stable
Multiplex detection of multiple protein targets without stripping and re-probing

Chemiluminescent detection
uses the horseradish peroxidase (HRP) enzyme and a luminescent substrate.
• Enzymatic reaction produces light that is detected by film exposure, or digital imaging with CCD camera
• Multiple exposures typically required to capture optimal signals and avoid signal saturation
• Very sensitive
• Cannot be multiplexed
• May not be quantitative

Colorimetric detection uses the alkaline phosphatase enzyme.
• Enzyme converts a soluble chromogenic substrate to a colored, insoluble product that precipitates onto the membrane and produces colored bands
• Development of the blot is stopped by washing away the soluble substrate
• Simple and cost-effective
• Limited sensitivity

Comparison of Chemiluminescence and Infrared Fluorescence
Detection for Western Blotting
Chemiluminescence IR Fluorescence
Sensitivity +++ +++
Linear Dynamic Range 10-50 fold >4000 fold
Multiplex Detection No Yes
Signal Stability Hours Months – Years
Enzyme Conjugate HRP
Substrate Luminol-based None Needed
Detection/Documentation Film Exposure/Digital Imaging Digital Imaging
Membrane Compatibility Nitrocellulose or PVDF Nitrocellulose or PVDF

Monitor Protein Levels and Phosphorylation with Quantitative Multiplexed Western Blots

Molecular profiling reveals diversity of stress signal transduction cascades in highly penetrant Alzheimer’s disease human skin fibroblasts.

Mendonsa, G., et al. PLoS ONE 4(2): e4655. doi:10.1371/journal.pone.0004655

Aberrant signal transduction is associated with Alzheimer’s disease (AD). In skin fibroblasts of AD patients, exaggerated signal transduction occurs in response to bradykinin (BK), an inflammatory neuropeptide. BK-induced PKC signaling causes stimulation of tau phosphorylation on serine residues in AD fibroblasts, but not in normal skin fibroblasts. Quantitative Western blotting with multiplex fluorescent detection (Odyssey Imager; LI-COR Biosciences) was used to monitor protein levels and phosphorylation.

To explore the roles of inflammatory and oxidative stress in AD pathology, this study profiled the effects of these stresses on MAPK signaling cascades in human skin fibroblasts of familial AD patients. AD fibroblasts of different genetic origins express presenilin (PS-1 or PS-2) mutated at a variety of sites. These mutations caused diverse responses to stress induced by BK or H2O2, with unique profiles of stress-induced MAPK activation, caspase-3 cleavage, and survival pathway activation. These results indicate that AD research must consider a broad spectrum of inflammatory, oxidative, and other stress factors and intracellular signaling responses.
Reduced ERK activation in PS-1 (M146L) Alzheimer's disease fibroblasts stimulated with bradykinin.

Figure 1. Reduced ERK activation in PS-1 (M146L) Alzheimer’s disease fibroblasts stimulated with bradykinin (BK). These fibroblasts carry a mutation in presenilin-1 associated with aberrant signaling. Mutant and control human skin fibroblasts were treated with 250 nM BK and immunoblotted for active and total ERK. Odyssey Imager was used, and fold activation was quantified. Total ERK is shown in green, and phospho-ERK in red; overlapping signals (active ERK) are shown in yellow. ERK activation was greatly reduced in PS-1 (M146L) AD fibroblasts. Graphs show mean + S.E. *p < 0.05 and **p < 0.005; n = 4. doi:10.1371/journal.pone.0004655

NEW! COX IV Primary Antibody Offers Normalization for Low-Expressing Proteins

COX IV Rabbit Monoclonal Primary Antibody, PN 926-42214The COX IV primary antibody can be used for detection of COX IV by Western blot, or as a normalization antibody when performing two-color detection. Its target molecular weight is 17 kDa. Detection of this primary antibody can be achieved with IRDye® Goat Anti-Rabbit or IRDye Donkey Anti-Rabbit secondary antibodies. LI-COR® also carries beta-actin, alpha-tubulin, and beta-tubulin primary antibodies for normalization when performing quantitative Western blots or In-Cell Western Assays.

Western Blot Linearity of COX IV vs. Actin in Cell Lysate

Figure 1. Linearity comparison of COX IV rabbit monoclonal primary antibody (P/N 926-42214) to β-Actin rabbit monoclonal (P/N 926-42210). Primary antibodies were compared by Western blot and detected with IRDye 800CW Goat anti-Rabbit (P/N 926-32211). The COX IV antibody can be used as a mitochondrial loading control and a loading control for normalizing low expressing target proteins. This COX IV primary antibody remains linear with increasing concentrations of lysate, making it ideal for normalization.

COX IV Rabbit Monoclonal Antibody Pack Insert

Now Use the Same Primary Antibody for Immunoprecipitation AND Western Blotting!

Quick Western Kit - IRDye 680RD, PN 926-68100

LI-COR® has just rolled out a new way that the recently-released Quick Western Kit – IRDye® 680RD (see Would you like to save at least 90 minutes the next time you do a Western blot?) can be used in your research.

Not only can the Quick Western Kit reduce Western blotting time by 90 min, the kit ALSO serves as a detection solution for post-immunoprecipitation samples by Western blot because it does not bind to denatured mouse monoclonal or rabbit monoclonal antibodies. The key benefit is the ability to use the same antibody for immunoprecipitation and post-immunoprecipitation detection by Western blot. Seriously, how cool is that!!??!!

Using the same primary antibody for IP and Western blotting with the Quick Western Kit

Figure 1. A431 cell lysates were immunoprecipitated overnight with a monoclonal antibody against p53. The resulting immunoprecipitates were separated by SDS-PAGE. Lane 1: Negative IP control; Lane 2: Test sample ; Lane 3: A431 cell lysate positive control. Western blotting was performed using the same p53 monoclonal antibody and incubated with IRDye 680RD Immunoprecipitation Detection Reagent.

Protocol: Detection of post-immunoprecipitation proteins by Western blot using the Quick Western Kit – IRDye 680RD

For more information, visit our website. Here’s the kit pack insert. To order this product (online ordering available in select countries), go to our ecommerce site.