Is Research Funding an Issue in Your Lab?

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NIH Funding Graph smallerIs research funding a main concern at your institution? In a study of 3700 researchers by the American Society for Biochemistry and Molecular Biology, “68% of respondents do not have the funds to expand their research operations.” Furthermore, “65% of respondents have had difficulties receiving funding.” This is an alarming number for the research community today.

Funding has been on the decline for some time now (see chart below), especially after the 2008 recession and the NIH sequester in 2013. In 2013, the NIH handed out “approximately 640 fewer research project grants compared to FY 2012.”

As budgets are tightened across the board, funding in general may be an issue in your lab. Besides funding to back research projects, faculty and researchers need reliable instrumentation in their labs to ensure reproducible, consistent results.

How will your institution remain equipped in an ever-increasing competitive environment? The LI-COR SURG Program** could help. The SURG – Science Undergraduate Research Grant – Program is designed for faculty researchers and their students to gain access to cutting edge life science technology. If students are learning Western blotting or gel imaging techniques, this grant program could be a perfect fit.

Odyssey Fc smallerLI-COR SURG grants are a 40% match from LI-COR. The process takes ten minutes to apply. We also provide suggestions on how to obtain the other 60% from funding and grant sources.

There’s no guarantee funding will increase in the future. This program could help ensure your research is supported by superior digital imaging technology. Check out the SURG Program** offered by LI-COR Biosciences if you’re interested in learning more. Here’s more information on the Odyssey® Fc Imaging System – LI-COR’s digital imaging solution offered through the SURG program.

** The LI-COR SURG Program is valid in the US and Puerto Rico only.

Studying Colon Cancer? Use the C-DiGit® Scanner for Western Blots.

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Cortactin (CTTN) is a substrate of Src tyrosine kinase involved in actin dynamics, and is overexpressed in several cancers. Phosphorylated cortactin (pTyr421) is required for cancer cell motility and invasion. This study demonstrates elevated expression of pTyr421-CTTN in primary colorectal tumors, with no change in mRNA levels. Curcumin (a natural compound derived from the spice turmeric) reduced association of CTTN with plasma membrane fractions in surface biotinylation, mass spectrometry, and Western blot experiments. Curcumin also decreased pTyr421-CTTN levels in certain cell lines.

Western blot analysis of cortactin, actin and GAPDH proteins

Figure 1. Western blot analysis of cortactin, actin and GAPDH proteins from DMSO and curcumin treated cell fractions of HCT116 cells. Total cell lysates were used to represent total protein input. Cytosolic and cytoskeletal proteins were extracted using Cell Fractionation kit (Cell Signaling, MA) and quantification of the blots are summarized in graphs. The images were scanned using C-Digit and quantified using Image Studio Digits (LI-COR Biosciences, NE). The data are expressed as a ratio to total protein (mean ± SD). * p<0.05 DMSO vs. curcumin; Student’s T-test. All images are representative of three independent experiments.

Quantitative chemiluminescent Westerns (using the LI-COR® C-DiGit Blot Scanner and SuperSignal® West Pico substrate) showed that curcumin treatment reduced CTTN levels in cytoskeletal fractions, and increased cytoplasmic localization. In Western blotting and immunofluorescent microscopy studies, curcumin induced dephosphorylation of cortactin by activation of the PTPN1 protein tyrosine phosphatase. Western blotting demonstrated that biotinylated curcumin directly binds to PTPN1, and that curcumin blocks the interaction between CTTN and p120 catenin. Curcumin inhibits cell migration in colon cancer cells overexpressing CTTN, and it holds promise as a colon cancer therapeutic.

Reference:

pTyr421 cortactin is overexpressed in colon cancer and is dephosphorylated by curcumin: involvement of non-receptor type 1 protein tyrosine phosphatase (PTPN1)
VM Radhakrishnan, P Kojs, G Young, R Ramalingam, B Jagadish, EA Mash, JD Martinez, FK Ghishan, PR Kiela
University of Arizona Health Sciences Center, Tucson, Arizona; Arizona Cancer Center, Tucson, AZ, USA
PLoS ONE 9(1): e85796 (2014). 10.1371/journal.pone.0085796

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

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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.

URGENT MESSAGE: Global Sources Report Imminent Pixel Shortage!

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Global pixel manufacturers report that a pixel shortage has reached crisis proportions.

Global Pixel Shortage
April 1, 2014, LINCOLN, NE, USA:

Sensel Raster, of the International Pixel Coalition (IPC), blames an increase in demand from biotech imaging applications. “These fancy scientists think pixels grow on trees!” laments Raster.

Just look at how this pixel shortage is affecting the LI-COR Biotechnology Website!!
April Fools Day Home Page

Learn more about this crisis.

Possible Cause 10 for Weak Chemiluminescent Western Blot Signals: Diluting Substrates

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WesternSure Chemiluminescent Western Blot ReagentsOkay, I know, research budget money is tight and you want to make your reagents stretch as far as possible, but it really not a good idea to dilute your chemiluminescent Western blotting substrate.

Why? It’s because the rate of reaction is determined by the ratio of enzyme to substrate. Diluting substrates will dramatically impact the overall generation of light. Then, you will have to repeat the experiment, and you end up using more substrate anyway!

Optimal Blot Unsatisfactory Blot
Images Optimal Western Blot - Substrate Not Diluted Unsatisfactory Chemiluminescent Western Blot - Substrate Diluted
Conditions:
Substrate SuperSignal® West Dura1 SuperSignal® West Dura1
Substrate NOT diluted. Substrate diluted 1:1 (in water)
Performance LOD – 1.25 µg LOD – 2.5 µg

1Comparable to WesternSure™ PREMIUM Chemiluminescent Substrate

So don’t skimp – use the substrate full strength the first time to ensure that you are seeing all of your protein bands. Or you might just have to repeat the experiment (and that will just cost you more time and money. . .)!

Here are the other nine possible causes of weak chemiluminescent Western blot signals:

Don’t Rush Substrate Incubation Time for Chemiluminescent Western Blots

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Substrate Incubation Time is Important!Five minutes can seem like a long time, especially when you are waiting to image your chemiluminescent Western blot. But it is really important that you follow the manufacturer’s recommendation for incubation time. Typically, this is five (5) minutes for optimal photon emission – for both film and digital imaging.

So, set the timer for 5 minutes, grab your iPhone® or iPod® – or the crossword, and relax until the buzzer goes off.

To test this, we imaged a chemiluminescent Western blot immediately after adding the chemiluminescent substrate and then imaged a blot where we waited 5 minutes – answered a few emails, looked at the news, and downloaded a new app – and THEN imaged the Western blot. As you can see, incubating allowed us to see more bands and gave much better Western blotting results.

Optimal Blot Unsatisfactory Blot
Images Optimal Blot - 5 Min Substrate Incubation Unsatisfactory Blot - No Incubation
Conditions:
Substrate SuperSignal® West Pico1 SuperSignal® West Pico1
Incubated for 5 minutes No incubation
Substrate at room temperature Substrate at room temperature
Performance LOD – 2.5 µg LOD – 5 µg

1Comparable to WesternSure™ ULTRA Chemiluminescent Substrate

So slow down, take a breath, and wait for your chemiluminescent Western blot substrate to incubate on your Western blot before imaging.

Here are some other blog posts on possible causes of weak chemiluminescent Western blot signals:

iPhone and iPod are all registered trademarks of Apple Inc.

Chemiluminescent Western Blot Substrate Temperature Affects Signal Strength on Western Blots

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The temperature at which a chemiluminescent Western blot substrate is used can affect the strength of the signal that is captured from Western blot images. Really?? Absolutely! This is because enzyme activity is greatly reduced when it is cold. The substrate needs to be equilibrated to room temperature for digital imaging. This is true with film as well, but there may be a period of time after adding substrate and exposing to film during which the substrate has had a chance to equilibrate to room temperature.

In the table below, we show data from an experiment in which we tested the affect of temperature on Western blotting signal. For one blot, SuperSignal® West Pico chemiluminescent substrate was used right out of the refrigerator – cold, 4 °C. For the other blot, the chemiluminescent Western blot substrate was allowed to come to room temperature before digital imaging. As you can see the signal difference is quite large.

Optimal Blot Unsatisfactory Blot
Images Optimal Blot when Substrate is at Room Temperature Unsatisfactory Blot when Substrate is Cold
Conditions:
Substrate SuperSignal® West Pico1 SuperSignal® West Pico1
Substrate at room temperature Substrate cold
Sensitivity Standard Standard
Performance Signal – 1,740 Signal – 200

1Comparable to WesternSure™ ULTRA Chemiluminescent Substrate

So make sure your substrate is at room temperature before using, especially when you are imaging with a digital imager!

Here are some other blog posts on possible causes of weak chemiluminescent Western blot signals:

Imager Sensitivity Settings May Affect Detection of Chemiluminescent Western Blot Signals

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Standard and High Sensitivity Settings on the C-DiGit
Standard and High Sensitivity Settings on the C-DiGit
Making sure that the sensitivity setting is optimal to capture the most signal from your chemiluminescent Western blot could be the difference between getting a good, strong signal or getting a signal that you can barely see. This is our possible cause 7 for weak chemiluminescent signals.

How can you avoid possible cause 7 for LI-COR chemiluminescent imagers? On the C-DiGit® Blot Scanner, use High Sensitivity setting (12-min scan) for more sensitive detection. On the Odyssey® Fc Dual-Mode Imaging System, use a longer integration time (up to 10 min). Why is this important? Well, digital imaging with the C-DiGit Blot Scanner or Odyssey Fc Imager will not generally reach a saturation point. Begin with a longer acquisition time to ensure best sensitivity, then optimize to shorter scan times.

In Table 1 below, we tested the performance differences of a Western blot detected with SuperSignal® West Dura on the C-DiGit Blot Scanner when the same blot is imaged on High Sensitivity (12 min scan) versus Standard Sensitivity (6 min scan). As you can see, the longer scan time and higher sensitivity make a big difference in the results.

Table 1 Optimal Blot Satisfactory Blot
Images Optimal Sensitivity Setting on C-DiGit Satisfactory Chemiluminescent Western Blot
Conditions: SuperSignal West Dura1 SuperSignal West Dura
Sensitivity High (12 min) Standard (6 min)
Performance Signal – 12,300 Signal – 5,030

1Comparable to WesternSure™ PREMIUM Chemiluminescent Substrate

So be sure to check your sensitivity settings before you scan!

Related posts:

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

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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

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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.