The Way Medical Film’s Future is Headed Will Keep You Up at Night

C-DiGit System, Western Blotting

What is the future of medical film?

Film Imaging Examples for Photography, Dentistry, Medicine, and ResearchNearly a year ago we told you why film’s future availability and affordability are in jeopardy. Today, we are still seeing a decreased demand and reduced production volume of film. But there are additional concerns. The environment is suffering because of the hazardous chemical and medical waste produced from using film.

Here are some realities facing Western blotters who use medical film:

  • The federal Resource Conservation and Recovery Act (RCRA) sets regulations for hazardous waste handling and storage.
  • The RCRA has strict laws with authority from the EPA enforcing toxic chemical cleanup.
  • Developer solutions must be neutralized and flushed with large quantities of water to the sewer system.
  • Film sheets should be collected for silver recycling because silver is too toxic to go in landfills.

What are the implications?

stas quoteAs environmental concerns rise and the supply of film is threatened, the sustainability and future of film production are at risk. As a responsible research scientist, you are aware there are environmental considerations and financial incentives for ceasing film use and switching to digital imaging. Read about one researcher who has come to that realization.

What can you do?

Consider an environmentally-friendly Western blot imaging alternative, and:

  • Eliminate your use of medical film
  • Decrease your environmental impact
  • Implement a more sustainable Western blotting technique

c-digit small

Go to bed at night without worrying if you can afford your next box of film or if you are complying with environmental hazardous waste disposal regulations. Go digital.

Register to win a C-DiGit® Chemiluminescent Western Blot Scanner today.

Need to Strip Both PVDF and Nitrocellulose Membranes? Try NewBlot™ IR Stripping Buffer!

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NewBlot IR Stripping BufferDoes your lab have both researchers that use PVDF membranes and researchers that use nitrocellulose for infrared Western blots? Are you still spending your valuable time making homemade stripping buffer?

NewBlot™ IR Stripping Buffer to the rescue! NewBlot IR is the latest member of the LI-COR NewBlot Stripping Buffer family.

All NewBlot buffers can be used for stripping and reprobing infrared fluorescent Western blots, and there is a formulation that works for whichever membrane type (or types) you use.

What makes NewBlot IR unique?

  • It strips both membrane types so you won’t need to buy separate stripping buffers for nitrocellulose and PVDF.
  • It is the most affordable, saving you money to spend on other items for your lab.

This robust reagent removes primary and secondary antibodies while maintaining target antigen integrity for efficient reprobing and does not require hazardous shipping, unlike many other stripping buffers.

Below are data showing that you can indeed strip and reprobe three times and still detect your proteins of interest.
NewBlot IR Strip and Reprobe Image

Figure 1. Strip and reprobe nitrocellulose or PVDF membranes effectively with NewBlot IR Stripping Buffer. EGFR and phospho-ERK levels were compared in EGF-stimulated (+) and non-stimulated (-) A431 lysate (1 µg total protein). The membrane was probed with mouse anti-EGFR, mouse anti-pERK1/2, and β-tubulin rabbit polyclonal (LI-COR P/N 926-42211), then detected with IRDye 800CW Goat anti-Mouse (LI-COR P/N 926-32210) and IRDye 680RD Goat anti-Rabbit (LI-COR P/N 926-68071). The blot was scanned with an Odyssey® CLx Imaging System (Original blot). The blot was then stripped with NewBlot IR Stripping Buffer (LI-COR P/N 928-40028), and scanned again (Strip #1). The blot was detected with the same primary and secondary antibodies and scanned again (Reprobe #1). The process was repeated 2 more times (Strip and Reprobe #2-3). Image display settings for all stripped and reprobed images are identical to the original image.

To make sure you are successful when stripping and reprobing, here are some factors that can affect stripping efficiency:

  • Amount of time the blot is in the stripping buffer
  • Sample type and preparation
  • Blot handling conditions
  • Buffer concentration and temperature used for stripping

Save your valuable time and money! Order NewBlot IR Stripping Buffer today for your infrared fluorescent Western blot stripping and reprobing needs.

Need Pre-stained Protein Ladders for Visible and Near-Infrared Detection?

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chameleon largerIf you are doing Western blots, then you are most likely using a protein ladder. And, if you need pre-stained protein molecular weight ladders that you can see AND detect with near-infrared fluorescence, then you need Chameleon Pre-stained Protein Ladders. These new ladders are multi-colored for easy molecular weight identification. So, you can easily identify gel migration and protein size and orient your gel and membrane quickly.

And, you have choices!

  1. Try the Chameleon Duo Pre-stained Protein Ladder if you are doing two-color infrared fluorescent detection. This Chameleon Ladder is a pre-mixed protein MW marker format– ready to use!
  2. Or, if you would like to mix your own to customize fluorescence intensities and are doing multiplex detection, try the Chameleon Kit Pre-stained Protein Ladder (includes 250 μL each of the Chameleon 700 and the Chameleon 800 Pre-stained Protein Ladders).
  3. Of course, if you are doing just one-channel analysis of your Western blot, you can try the
    1. Chameleon 700 Pre-stained Protein Ladder – for 700nm channel analysis.

      2. Or, try the

    2. Chameleon 800 Pre-stained Protein Ladder for analysis in the 800nm channel.

chameleon all
So, you choose! Then, experience the sharp, vibrant colors of LI-COR Chameleon Pre-stained Protein Ladders in your near-infrared fluorescent Western blot applications.

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!

C-DiGit System, , , , , ,

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: