Article Category: Reagents

Avoid Milk Blocking Buffer – Use NEW! Odyssey® Blocking Buffer (TBS)

Odyssey Blocking Buffer (TBS)

In previous posts, we’ve talked about Western blot blocking buffers and how important it is to optimize your blocking conditions to get the best results. As many of Western blot users do, you may just routinely use homemade TBS-milk blocking buffer. It’s inexpensive, and it does the job. . . well, most of the time. . .

What you may not know is using milk blocking buffer can cause issues with certain targets. This may give you the wrong information about the presence or the amount of your target. One good way to determine which blocking buffer system to use is to check to see what the primary antibody vendor recommends. Most recommend TBS-based buffer systems. If the primary antibody requires a TBS-based buffer system, we recommend new Odyssey™ Blocking Buffer (TBS).

When should you avoid milk blocking buffer?

  • When using anti-goat secondary antibodies.
    • Reason: Milk contains bovine IgG. Anti-goat secondary antibodies may recognize bovine IgG, resulting in high background.
  • When detecting phosphorylated proteins.
    • Reason: Milk contains phosphorylated proteins, which may result in low to no signal and high background.
  • When using streptavidin-biotin detection systems.
    • Reason: Milk contains endogenous levels of biotin. Streptavidin will detect this, resulting in high background.

OBB TBS and milkHere are the results of an experiment evaluating the use of milk and Odyssey Blocking Buffer (TBS). As you can see, milk masked the detection of this protein and is not a good blocking buffer choice.

Figure 1. Effect of various blocking agents on detection of pAkt and total Akt in Jurkat lysate after stimulation by calyculin A. Total and phosphorylated Akt were detected in calyculin A-stimulated (+) and non-stimulated (-) Jurkat lysate at 10 µg; 5 µg; and 2.5 µg/well. Blots were probed with pAkt Rabbit mAb (Santa Cruz P/N sc‑135650) and Akt mAb (CST P/N 2967) and detected with IRDye® 800CW Goat anti-Rabbit IgG (LI‑COR P/N 926-32211) and IRDye 680RD Goat anti-Mouse IgG (LI‑COR P/N 926‑68070); scanned on Odyssey® CLx (auto scan 700 & 800). pAkt (green) is only detected with Odyssey Blocking Buffer (TBS).

So be sure to optimize your Western blot blocking conditions! The time you spend finding the best blocker will be worth it – and save you from making the wrong conclusions about your experimental data in the future.

Find Your Perfect Match with IRDye® and VRDye™ Secondary Antibodies!

Your Perfect Match - LI-COR Secondary Antibodies

Straight from Cupid for specific bonding!

IRDye® and VRDye™ Secondary Antibodies are the perfect match for your research! We offer highly cross-adsorbed secondary antibodies conjugated to:

  • IRDye 800CW
    • Including IgG1, IgG2a, and IgG2b Subclass Specific and Goat anti-Mouse IgM (μ chain specific) Secondaries
  • IRDye 680RD
    • Including Goat anti-Mouse IgM (μ chain specific) Secondary Antibody
  • IRDye 680LT
    • Including IgG1, IgG2a, and IgG2b Subclass Specific and Goat anti-Mouse IgM (μ chain specific) Secondaries
  • IRDye 650
  • VRDye 549
  • VRDye 490

To find which LI-COR secondary antibody is the perfect match for your experimental needs, be sure to review the specific applications for which each dye-conjugated secondary antibody is recommended. We also have Protein Labeling Kits in various dye ‘flavors’. Protein labeling kits are cost-effective alternatives to more expensive custom antibody labeling services:

IF, however, you find you do have a special labeling or synthesis need, LI-COR now offers a variety of custom labeling and synthesis services that go beyond our basic offerings. Based on our many years of experience in dye conjugation, our custom services provide a unique solution for most custom needs. We also offer protocol development for In-Cell Western™ Assays, Western blotting, and other applications. The newest offering from our Custom Services group is Reactive Oxygen Species probes.


Happy Valentine’s Day from LI-COR!

Annotate Visible Protein Ladders on Chemiluminescent Westerns with the WesternSure™ Pen

Demonstrating the WesternSure PenIf you doing chemiluminescent Western blots, and are imaging either with film or with a digital imager, the WesternSure™ Pen can be a very useful addition to your experimental process. This newest member of the LI-COR WesternSure chemiluminescent reagent line can be used to annotate visible protein ladders prior to chemiluminescent Western blot detection.

The pen is optimized for detection using the C-DiGit® Blot Scanner or the Odyssey® Fc Imaging System, and is suitable for use with film or other imaging systems. The WesternSure Pen is a unique marker that delivers an ink which emits light when incubated with commonly-used chemiluminescent substrates, including WesternSure PREMIUM Chemiluminescent Substrate. The ink is faintly visible for easy identification of marked membranes.

Here are a few tips to get the best performance from your WesternSure Pen:

  • Lightly touching the pen to the membrane should be enough to transfer ink to the membrane.
  • Do not push down on the nib so hard that it creates an uneven surface on the membrane.
  • Membranes may be annotated when damp after transfer, or when dry.
  • Annotated membranes may be stored dry at ambient temperature or 4 ºC for up to 1 week before starting the Western blot detection process.
  • If ink is not flowing smoothly onto a damp membrane, trace over the band until it is annotated to the desired effect.


Data using the WesternSure PenFigure 1. Chemiluminescent detection of visible protein standards. The WesternSure Pen (LI‑COR P/N 926‑91000) was used to mark the blue protein standards (panel A) for chemiluminescent Western blot detection. The blot was exposed to WesternSure PREMIUM chemiluminescent substrate and imaged on Odyssey Fc Imaging System (panel B).

If you would like some tips on how to troubleshoot chemiluminescent Western blots, read Good Westerns Gone Bad – Maximizing Sensitivity on Chemiluminescent Western Blots.

In the US, to order the WesternSure Pen online. For order inquiries outside the US, please contact your local sales office.

Troubleshooting Chemiluminescent Western Blots: Possible Cause 4 for Weak Signals – Blot Processing

Sometimes life in the lab gets crazy, right? You are finishing a Western blot and you realize that you are supposed to be at an important lecture across campus in 10 min!! Or, your spouse calls to say that one of the kids needs to be picked up as soon as possible. Yikes! The challenge is that blots should be processed and detected on the same day. And, the secondary antibody should be incubated the day of imaging and fresh substrate added just before imaging. Is it that important to your results? Yes, it is and just to prove it, we did a few experiments.

In Table 1, we studied performance differences when the same blot is imaged immediately after processing vs. stored overnight dry and then imaged. In Table 2, we looked at performance differences when the same blot is imaged immediately after processing vs. stored overnight wet and then imaged. Blots in both tables were all imaged on the C-DiGit® Blot Scanner. (And, all images are normalized to the Lookup Tables (LUT) of the respective optimal blot.)

For both experiments, you can see that saving the blot to image the next day is not a very good choice. This is because the secondary antibody and/or the chemiluminescent Western blot substrate is not stable enough for acceptable photon emission when digitally images after the day it is applied.

Table 1 Optimal Blot Unsatisfactory Blot Unsatisfactory Blot
Images Optimal Chemiluminescent Western Blot Unsatisfactory Chemiluminescent Western Blot Unsatisfactory Chemiluminescent Western Blot
Conditions:
Substrate SuperSignal® West Dura1 SuperSignal West Dura1 SuperSignal West Dura1
Processing Time Same Day Next Day Next Day
Detection Process HRP secondary incubated, washed, and substrate added immediately before imaging. HRP secondary incubated, washed, and substrate added day before imaging. HRP secondary incubated, washed, and substrate added day before imaging, then re-incubated with HRP secondary and substrate added immediately before imaging.
Storage Conditions Blot stored overnight dry, at room temperature Blot stored overnight dry, at room temperature
Performance LOD – 640 ng LOD – None detected LOD – 1.25 μg
Table 2 Optimal Blot Unsatisfactory Blot Unsatisfactory Blot
Images Optimal Chemiluminescent Western Blot Unsatisfactory Optimal Chemiluminescent Western Blot Unsatisfactory Optimal Chemiluminescent Western Blot
Conditions:
Substrate SuperSignal® West Dura1 SuperSignal West Dura1 SuperSignal West Dura1
Process Time Same day Next day Next day
Detection Process HRP secondary incubated, washed, and substrate added immediately before imaging. HRP secondary incubated, washed, and substrate added day before imaging. HRP secondary incubated, washed, and substrate added day before imaging, then re-incubated with HRP secondary and substrate added immediately before imaging.
Storage Conditions Blot stored overnight wet in PBS, at room temperature Blot stored overnight wet in PBS, at room temperature
Performance LOD – 640 ng LOD – None detected LOD – 1.25 μg

1SuperSignal West Dura results are comparable to those obtained with WesternSure™ PREMIUM Chemiluminescent Substrate.

For more hints and tips, stay tuned to future blog posts. And if you would like to try some FREE Western Blot Analysis Software, download Image Studio Lite today!

Related posts:

Use Near-Infrared Fluorescent Probes for Pharmacokinetics and Biodistribution Studies

In Vivo Imaging with NIR Fluorescent ProbesNon-invasive preclinical imaging methods are critical for development of imaging agents and targeted therapeutics. Pharmacokinetics is the study of what the body does to a drug with respect to biodistribution and clearance. Traditionally-used radiolabeled probes have limitations such as cost, access, and safety. Near-infrared (NIR) fluorescence imaging offers a powerful alternative to radiolabeled probes for pharmacokinetics and biodistribution studies. NIR fluorescent optical imaging agents can be used to image the whole animal over time. And, more than one agent can be tracked in the same animal if each agent is labeled with a spectrally-distinct fluorophore.

In this webinar, Dr Amy Geschwender examines several case studies from the literature, and discusses:

  • Why NIR fluorescent probes are widely used for in vivo imaging
  • How fluorescence imaging of excised tissues and tissue sections is used to examine biodistribution in more detail
  • How to measure serum half-life and % injected dose per gram with NIR fluorescent probes

This webinar features data from the Pearl® Small Animal Imaging System, which was recently honored by Frost & Sullivan, in addition to advancements in NIR technology. Click here to learn more about this award.

Visit our website to learn more about BrightSite™ Optical Imaging Agents and IRDye® infrared dyes that can be used for your pharmacokinetic and biodistribution studies.

Weak Signals on Chemiluminescent Western Blots: Possible Cause 1 – Substrate Rate of Reaction

Optimal Chemiluminescent Western BlotAre you seeing weak signal in your chemiluminescent Western blot data? As we pointed out in a previous blog post, there are 10 possible reasons why this may be happening. Here is the first in our series on the causes and possible solutions/prevention measures you can try to get the best Western blot imaging data you can from your digital imager! We used our Odyssey® Fc Dual-Mode Imaging System and the newest member of our imaging family, the C-DiGit® Blot Scanner, in these studies.

Possible cause 1: Substrate does not have a fast enough rate of reaction (e.g., SuperSignal® West Pico)

Solution: Use WesternSure™ PREMIUM or SuperSignal West Femto substrates

Why this matters: Different substrates have different rates of reaction. Some are developed to give off a lot of light quickly; others give off small amounts of light over longer periods of time. An alternate substrate may be required for digital imaging when imaging blots with low protein abundance.

Performance differences of three different substrate classifications using C-DiGit® Blot Scanner. All images are normalized to the Lookup Table (LUT) settings of the optimal blot for accurate visual comparison. (Learn more about easy-to-use Image Studio Software.)

Optimal Blot Satisfactory Blot Unsatisfactory Blot
Images Optimal Chemiluminescent Western Blot Satisfactory Chemiluminescent Western Blot Unsatisfactory Chemiluminescent Western Blot
Conditions:
Substrate SuperSignal West Femto SuperSignal West Dura1 SuperSignal West Pico2
Substrate Volume 3.0 mL substrate 3.0 mL substrate 3.0 mL substrate
Imaging Method
  • Substrate placed directly on C-DiGit Blot Scanner glass surface.
  • Membrane placed on substrate, 1-ply sheet protector on top, incubate 5 min.
  • Substrate placed directly on C-DiGit Blot Scanner glass surface.
  • Membrane placed on substrate, 1-ply sheet protector on top, incubate 5 min.
  • Substrate placed directly on C-DiGit Blot Scanner glass surface.
  • Membrane placed on substrate, 1-ply sheet protector on top, incubate 5 min.
  • Scan Setting High High High
    Performance LOD – 78 ng LOD – 312 ng LOD – 2.5 μg

    1Comparable to WesternSure PREMIUM Chemiluminescent Substrate
    2Comparable to WesternSure ULTRA Chemiluminescent Substrate

    If you want to read ahead and find out ways to eliminate or avoid the other 9 causes of weak signals on chemiluminescent Western blots, read Good Westerns Gone Bad: Maximizing Sensitivity on Chemiluminescent Western Blots. Otherwise, stay tuned for more posts right here!

    10 Possible Causes of Weak Signals on Chemiluminescent Western Blot Images

    Weak Signals on Chemiluminescent Western BlotsAre you seeing weaker than expected (hoped for. . .) signal on your chemiluminescent Western blot images with your digital imager? Not sure what could be causing this? Well, here is a list of 10 possible reasons why you might be seeing weak signals in chemiluminescent Western blot data:

    1. Thechemiluminescent substrate does not have a fast enough rate of reaction.
    2. Not enough substrate was added to the blot.
    3. Membrane was placed on the detection system incorrectly.
    4. Blot was not detected or processed on the same day it was imaged.
    5. Blot was not kept uniformly wet during the entire image acquisition.
    6. Blot was exposed to film BEFORE imaging on a digital imager.
    7. Blot was imaged using incorrect sensitivity setting (learn about the easy-to-use Image Studio Software. Try FREE Image Studio Lite Western Blot Analysis Software to see just how easy it is!)
    8. Chemiluminescent substrate was too cold.
    9. Chemiluminescent substrate was not incubated for 5 minutes.
    10. Substrate was diluted.

    Hum, that’s quite a list! For details on ways to eliminate or avoid these causes and get great results with your chemiluminescent Western blots, read Good Westerns Gone Bad: Maximizing Sensitivity on Chemiluminescent Western Blots.

    Use NEW! VRDye™ Secondary Antibodies to Correlate Near-Infrared Application Data with Microscopy and Flow Cytometry Data

    VRDye Secondary Antibody IconsLI-COR is expanding its portfolio of reagents by offering VRDye™ 490, VRDye 549, and IRDye® 650 dye-labeled secondary antibodies and protein labeling kits. These new secondaries can be used for for a variety of applications, including immunofluorescence microscopy and flow cytometry. Just like our IRDye dye-labeled secondary antibodies, these new visible fluorescence antibodies are highly cross-adsorbed. The dyes are conjugated to the same antibodies as the existing IRDye secondary antibodies, which are used for Western blotting and In-Cell Western™ Assay applications. This gives researchers the ability to correlate microscopy and flow data with Western blot and cell-based assay data. The VRDye secondary antibodies are suitable for multiplex experiments when combined with other secondary antibodies labeled with proper fluorescent dyes and using instrumentation with appropriate excitation and detection capabilities.

    Immunofluorescence staining of tubulin protein in HeLa cells.

    Figure 1. Immunofluorescence staining of tubulin protein in HeLa cells. Cells were cultured on cover slips. After fixation and permeabilization, cells were incubated with rabbit anti-tubulin mAb (CST), followed by VRDye™ 490 Goat anti-Rabbit IgG (LI-COR P/N 926-49020). Nuclei were stained with DAPI. Image acquired with Olympus IX81 microscope.

    Immunohistochemistry staining of EGFR protein on F98-EGFR tumor slides.

    Figure 2. Immunohistochemistry staining of EGFR protein on F98-EGFR tumor slides. F98-EGFR tumors were snap-frozen in O.C.T. ™ compound and sectioned at 4-µm thickness. After fixation and permeabilization, cells were incubated with rabbit anti-EGFR mAb (CST), followed by detection with VRDye™ 549 Goat anti-Rabbit IgG (LI-COR P/N 926-54020). DAPI was used to stain the nuclei. Image acquired on Olympus IX81 microscope.

    In addition, many researchers use labeled primary antibodies for flow cytometry. LI-COR now offers visible fluorescent dye protein labeling kits that are ideal for customers who need to label custom monoclonal antibodies for this application.

    Visit our website for more information on these new visible fluorescence antibodies and protein labeling kits or to order them for your research.

    Easily Annotate Protein Molecular Weight Ladders on Chemiluminescent Western Blots

    Glow-Writer Pen for Chemiluminescent WesternsThe Glow-Writer pen (LI-COR P/N 926-90000) is a phosphorescent marker used to annotate visible protein molecular weight ladders for chemiluminescent Western blot detection. The procedure described below has been optimized for detection using the C-DiGit® Blot Scanner or the Odyssey® Fc Imaging System. The Glow-Writer pen may be suitable for use with film or other imaging systems; however, further optimization may be necessary.

    Here are the easy step-by-step instructions on how to use the Glow-Writer Pen. Please read all steps below prior to use. The most effective time to annotate the blot is immediately before the addition of substrate.

    1. Shake the pen vigorously for 30 – 45 seconds prior to each use.
    2. On a paper towel, press down on the pen’s nib. Keep the nib pushed in until you see green ink saturate the nib. This may take 30 seconds to a minute.
    3. Before attempting to write on a blot, ensure that the pen writes smoothly without having to push down on the nib.
      • NOTE: It is best to test the pen on a piece of lab tape or parafilm just before writing on the blot to ensure that the pen is writing smoothly.
    4. Trace the visible ladder on the membrane. It may be necessary to wick excess fluid off the membrane using a tissue prior to annotation. Lightly touching the pen to the membrane should be enough to transfer ink to the membrane.
      • NOTE: Do not push down on the nib so hard that it enters the pen chamber while the pen is on the membrane. Doing so will flood the membrane with ink.
    5. Substrate can be added immediately after writing on the membrane with the Glow-Writer pen.
    6. After substrate incubation, expose the membrane to light for 30 – 60 seconds. Leaving the membrane on the bench top should be sufficient light exposure to “excite” the ink. If bands are not bright enough, closer exposure to a light source may increase band brightness. Ink glows for approximately 5 minutes.
    7. Re-expose to light if blots are imaged again.

    Get a Glow-Writer Pen today so that you, too, can easily annotate protein molecular weight ladders and ensure that you can detect this important size on film or chemiluminescent Western blot imaging systems.

    Multiplex Western Blotting System Turbo-Charges Western Blot Results Output

    Example of Multiplexed Western Blot using the MPX Blotting SystemMultiplexing 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:

    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.