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!

Coming in 2013! LI-COR® C-DiGit® System – Digital Film for Chemiluminescent Western Blot Imaging

Are you tired of waiting in line for the darkroom? Or spending your precious budget monies on all that film? OR having to do multiple exposures to get just the right image for publication? Then, you need DIGITAL FILM!

But what, you may be asking, is digital film? Well, that’s the LI-COR C-DiGit System! Image your chemiluminescent Western blot and get great images for publication the FIRST time!

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

Troubleshooting In-Gel Westerns – Where’s the Signal?

In-Gel Western with Two-Color DetectionOkay, so you’re doing an in-gel western because you have a hard-to-transfer target (say, a glycoprotein). And you are using near-infrared fluorescence detection because it gets rid of inconsistencies due to transfer (and with an Odyssey it’s really fast and efficient to image and analyze!)

You read the In-Gel Western troubleshooting blog from March 6, 2012, but right now, what you’re seeing, or, um, well, NOT seeing is a signal. Rats! Where IS it?

Well, here are some possible causes with ways to solve or prevent this from happening:

Not enough antibody.
— Increase amount of primary and/or secondary antibody. Extend primary antibody incubation to overnight at 4°C to increase signal.
— Remember that In-Gel detection is not as sensitive as blot detection; adjust sample loading and antibody concentrations accordingly.
Antibody dilution buffer is not optimal for your primary antibody.
— Try a different dilution buffer; this can significantly affect performance of some primary antibodies.
— Suggested buffers include 3-5% BSA, Odyssey Blocking Buffer (PBS), or Odyssey Blocking Buffer (TBS), and PBS or TBS (all with 0.1% Tween® 20). Other blockers (milk, casein, commercial blockers) and Tween 20 concentrations can also be tested.
Gel type is not optimal.
— Amresco NEXT gels or NuPAGE® Bis-Tris pre-cast gels are recommended for In-Gel detection. Other commercial gel sources and homemade gels can be used, but may show reduced sensitivity and require further optimization.
Antibody did not penetrate gel sufficiently or evenly.
— Acrylamide percentage was too high. Try a lower percentage or a gradient gel.
— Increase volume for antibody incubations so that gel is completely immersed in antibody solution.
— Make sure gel is adequately fixed. Some monoclonal antibodies may be sensitive to residual acid in the gel; in this situation, eliminate acetic acid from the fix or extend the water wash step.
Gel was left in isopropanol/acetic acid too long.
— This may cause protein to be lost from the gel. Fix for 15 minutes only.

Whew! Well, hopefully by using one of these tips, you are NOW seeing a signal from your protein. Stay tuned for more troubleshooting tips for near-infrared fluorescent In-Gel Westerns in future blogs!
In-Gel detection of Cytochrome P450 3A4 (CYP3A4).

In-Gel detection of Cytochrome P450 3A4 (CYP3A4). Fixed gel was probed with anti-CYP3A4 primary antibody and IRDye® 800 secondary antibody. The limit of detection is approximately 3 ng. Reprinted with permission from Theisen, M. J. and Chiu, M. L. LI-COR Biosciences (2004)

Digital Western Blots – Chemiluminescent Substrate Selection is Critical

Chemiluminescent Western BlotSo, you’ve selected your primary antibodies with care (see Know Thy Primary Antibody), and you’re using a great HRP-conjugated secondary antibodies. NOW – what about the chemiluminescent substrate?

Yes, I know, there are tons of different substrates and vendors out there – all claiming to be the best, right? So, how do you choose the right one for your chemiluminescence Western blot?

One thing to keep in mind is that in this wide variety of chemiluminescent substrates for HRP detection, there are some that are better suited for digital Western imaging than others. In general, choose a substrate with a faster rate of reaction for use with the Odyssey Fc Dual-Mode Chemiluminescent and IR Fluorescent Imaging System or other digital chemiluminescent imaging systems.

Some substrates that are designed for optimal performance on film may not be suitable for detection on a CCD-based imaging system. Try different substrates to find the one that gives the most desirable image. As you can see from the images below, the substrate you pick DOES make a difference! So choose carefully!

In the three images below, two-fold serial dilutions of HRP-conjugated secondary antibody (1 ng-1.25 fg) were spotted onto nitrocellulose using a slot blot apparatus. Blots were detected with various chemiluminescent substrates.

Chemiluminescent Substrate Comparison

Here are 2 documents with more troubleshooting information:

Updated February 18, 2015.

Detect Difficult Proteins More Easily with Near-Infrared In-Gel Westerns

Western blot detection of proteins requires separation of protein mixtures by electrophoresis, followed by transfer of the separated proteins to nitrocellulose or PVDF membranes for detection. In-Gel Western detection avoids transfer problems by directly detecting target proteins within the polyacrylamide gel matrix, using the Odyssey® CLx or Classic Infrared Imaging System or the Odyssey Fc Imaging System.

The Odyssey Infrared Imaging systems allow you to detect target proteins while still embedded in the gel – without transfer to a membrane – using near-infrared secondary antibodies, such as the LI-COR® IRDye Conjugates. Using near-infrared fluorescence detection methods for In-Gel Westerns makes this a powerful technique. It saves time, reduces cost, and eliminates the variables introduced by the transfer step or subsequent blocking of the membrane. In-Gel Western detection can be performed with standard Odyssey reagents – no special kit is required.

Comparing Odyssey Infrared Detection of In-Gel Westerns vs. Chemiluminescence Detection

Figure 1. Sensitivity of Odyssey infrared In-Gel Westerns is equal to or better than chemiluminescence. Beginning with 10 ng/lane (far left), two-fold serial dilutions of purified Transferrin were separated by electrophoresis on duplicate gels. In-Gel Westerns were detected with infrared fluorescence (top) and chemiluminescence on film (bottom). Odyssey detection outperformed chemiluminescence.

 

For more information, refer to Odyssey® In-Gel Western Detection Protocol and the In-Gel Western application pages.