Requirements for Internal Loading Controls

Western blots are packed with potential sources of variability. Variability that isn’t accounted for limits reproducibility and threatens your chances for publication-quality data. Normalization corrects for variability introduced during the process of Western blotting.

So what should you do to get more reproducible data? Use an internal loading control for each blot. Internal loading controls are endogenous sample proteins that are stably expressed and unaffected by experimental conditions.

Requirements for an Effective Internal Loading Control:

  • Linear, proportional response. Signal intensity of the internal control should accurately reflect sample con¬centration and abundance of loading control over a wide range.
  • Low biological variability. Your experimental treatments should not affect the expression of your internal loading control. For example, expression of some housekeeping proteins may vary in response to experimental conditions.
  • Corrects for variation at all stages of immunoblotting. Your internal control should correct for variation that occurs throughout the Western blot process, including gel loading and transfer.
  • Compatible with immunodetection. The strategy you choose shouldn’t interfere with effective down¬stream detection of your target proteins.

For more information about internal loading controls, check out the full review article:
Western Blot Normalization: Challenges and Considerations for Quantitative Analysis

What Factors Affect Normalization?

Do you know what factors affect normalization? Routine steps in the Western blotting process such as sample preparation, sample loading, and the detection of multiple proteins can introduce unwanted variability. You should plan to reduce error in every step of the Western blotting process. Without planning, you might get pseudo-quantitative results that don’t reflect the biology of your samples.

Sample Preparation

Blog - Sample prepThe way you prepare your samples can significantly change the results of your experiment. Even small changes in plating, cell lysis, reagent volume, and other technical details can have a surprising impact.

For example, how you lyse your cells affects protein extraction, solubilization, and modification status. The insoluble fraction may retain relevant proteins, affecting your quantitative analysis. Some experimental treatments shift fractions between soluble and insoluble.

For these reasons, it’s important to be consistent when preparing your samples. It’s also good practice to estimate the total protein concentration of each sample after preparation. Bradford, BCA, and Lowry assays are widely used to estimate the total protein concentration. Then it’s possible to adjust gel loading to the estimated protein concentration.

Sample Loading

Blog - sample loadingOverloaded gels create problems. Although strong bands may appear similar, the bands could be saturating either the membrane capacity or the dynamic range of detection. To avoid saturation and inaccurate results, run a standard curve with two-fold serial dilutions of cell lysate. You can then identify the linear range for each target protein.

Detection of Multiple Proteins

You may need to detect multiple proteins to compare relative protein levels, especially if you’re using a housekeeping protein or signaling protein to normalize. Stripping and reprobing is often used to compare different proteins on the same blot, but it can introduce error. Leftover antibodies from incomplete stripping result in artifacts. Overly harsh stripping may result in a loss of sample proteins from the membrane.

If, however, you use near-infrared fluorescent detection, there’s no need to strip and reprobe. Multiplexing is when you detect two different proteins with spectrally-distinct secondary antibodies. Multiplexing is convenient and saves time. It is also more accurate than stripping and reprobing, because no artifacts are introduced, and there’s no possibility for protein loss. With multiplexing, you can use co-migrating proteins, as well as easily identify antibody cross-reactivity.

For more details about factors that affect normalization, check out the full review article:
Western Blot Normalization: Challenges and Considerations for Quantitative Analysis

Understanding Western Blot Normalization

chess piece - kingResearchers rely on Western blotting to detect target proteins in complex samples. This trusted technique is widely used to compare relative protein levels.

However, variability can creep into your Western blots through differences in sample preparation, sample loading, and transfer from gel to membrane. That’s why normalization is important. Normalization is the process of using internal loading controls to mathematically correct for sample-to-sample variation. These internal loading controls verify whether or not samples are uniformly loaded across the gel, confirm consistent transfer from gel to membrane, and enable comparison of relative protein levels between samples.

Normalization is meant to correct for small variation between samples, and can’t completely remove variability. If large data corrections are applied, accuracy may be affected. Normalization is a strategy to apply throughout your experiment, rather than a last step in the protocol. The more sources of variability you can reduce or eliminate, the more reproducible your experiment will be.

The role of an internal loading control is always to confirm the changes you see on the blot reflect actual change in the biology of your samples. To demonstrate statistically significant changes in the abundance of target protein, you need a reliable normalization strategy that fits the context and biology of your experiment. Effective, carefully-planned normalization will more accurately reflect the amount of protein in each lane.

chess piece - bishopUnderstanding Western blot normalization will help you choose a strategy that fits the context and biology of your experiment.

This paper describes important considerations, strengths, and limits of commonly used normalization strategies:

Western Blot Normalization: Challenges and Considerations for Quantitative Analysis

Are Western Blot Results Misrepresented by Film and Photochemistry?

Although most researchers have used film to document Western blots, many may be unfamiliar with the photochemical process that creates a visible image on a sheet of x-ray film. Because this process affects data output, it is important to understand how chemiluminescent signals are recorded by film – particularly if the results will be quantified by densitometry.1

What happens when you expose a Western blot to film?

X-ray film is coated with a photographic emulsion that contains light-sensitive silver grains. Photons of light from the chemiluminescent reaction activate individual silver grains, which are then converted to black metallic silver to create a visible film image. Within the film’s linear response range, your results are proportional to light intensity and duration; this is called the Reciprocity Law.

What goes wrong during film exposure?

Film’s linear response range is extremely narrow (1.0 – 1.5 logs). Above and below that narrow range, “reciprocity failure” occurs – and your bands won’t be proportional to the light produced by the chemiluminescent reaction. It’s important to know that both strong and faint signals are not accurately detected by film, which compromises the accuracy of your densitometry results.
film vs photchem image

How does reciprocity failure affect your densitometry and data analysis?

In this example, film response is only linear between 0.1 ng and 1.56 ng. Above 1.56 ng, bands visually appear stronger on film, but signals are not accurately recorded due to high intensity reciprocity failure.

film vs photochem image 2
RESULT: Strong bands are underestimated by densitometry. Film’s limited dynamic range interferes with accurate detection of strong signals.

Improve the accuracy of your results

The photochemistry of film causes a non-linear response of film to faint and strong signals (reciprocity failure). Saturation of strong signals and under-representation of faint signals means that accurate densitometry is severely limited by film’s shortcomings. When you switch to a digital imager, you will get more accurate results. Read the full paper to learn about all the variables that affect accurate quantification:

  • Enzyme/substrate kinetics and changes in substrate availability
  • Limitations of film exposure and digitization methods
  • Difficulty determining the saturation point of strong signals

Read the full study: Chemiluminescent Westerns: How film and photochemistry affect experimental results

Reference:
1. Baskin, DG and WL Stahl. Fundamentals of quantitative autoradiography by computer densitometry for in situ hybridization, with emphasis on 33P.
41(12):1767-76 (1993).

Is Your Chemiluminescent Western Blot Imaging Method a Source of Error and Variability?

Chemiluminescence is a dynamic, enzymatic process that introduces variability and error in your Western blot experiments. It’s often difficult to find the “best” exposure, and the need for multiple exposures limits the reproducibility of your results.

Variability and error are introduced because:

  • Chemiluminescent reaction changes constantly.
    The “best” exposure time is a moving target, so you must optimize and double-check every experiment.
  • Multiple exposures are required.
    Common detection methods cannot accurately capture both faint and strong signals at once, without signal saturation.

 

Usable Data for Each Detection Method

Film Imager B Odyssey® Fc Imager
film usable range imager b usable range odyssey fc usable range
RESULT: Exposure time dramatically affects data output. Multiple exposures are required to detect strong and faint signals. Signal saturation cannot be determined visually. RESULT: Multiple exposures are required to capture the full range of data. Strong signals are saturated (shown in blue). RESULT: Multiple exposures are not required, because all exposure times yield consistent results. All data are captured in a single exposure without saturation.

In the figure above, film was compared with a conventional, commercially-available CCD imager (Imager B), and the Odyssey Fc imager. To eliminate variability introduced by blotting and chemiluminescent detection chemistry, a Harta luminometer reference plate (standardized light source) was used in place of a Western blot.

CONCLUSIONS
The Odyssey Fc imager outperformed both film and Imager B. All signals, from faintest to strongest, were detected – regardless of exposure time in a single exposure. No signal saturation occurred and all signals could be quantified. With film and Imager B, however, longer exposures are needed to detect faint signals. In addition, stronger signals become saturated and cannot be quantified.

Choosing the Odyssey Fc Imaging System as your imaging method reduces variability and error in chemiluminescent Western blotting by giving you:

  • All your data in a single exposure
  • More reproducible results
  • Simplified data analysis

Read the full study to learn:

  • How chemiluminescence detection introduces variability and error
  • How you can improve the reproducibility of your Western blot data

Film and CCD Imaging of Western Blots: Exposure Time, Signal Saturation, and Linear Dynamic Range

No-Hassle Near-Infrared Fluorescent and Bioluminescent Optical Imaging

Pearl Trilogy Workstation
The Pearl® Trilogy Small Animal Imaging System is a simple, economical way for your lab to begin in vivo optical imaging. Starting at $65,000 (US List), the Pearl Trilogy offers affordable small animal near-infrared fluorescent and bioluminescent optical imaging.

Combine the Pearl Trilogy with the Odyssey® CLx Imager and create a complete workstation that allows you to go from in vitro to in vivo to ex vivo using the same trusted technology and reagents.

The revolutionary FieldBrite™ Xi2 technology approach to imaging in the Pearl Trilogy Imager allows you to detect smaller and deeper targets accurately in a single acquisition. You don’t need to worry about saturation or having to adjust images.

FieldBrite Xi2 is specifically optimized for small animal imaging, ensuring that you get the highest quality data possible. FieldBrite Xi2 technology offers:

  • Uniform Illumination
  • Excellent Sensitivity
  • Wide Dynamic Range

Bioluminescent Optical Imaging

Figure 1. Bioluminescent detection of subcutaneous 4175 (LM2) luc + human triple negative breast cancer cell line in athymic NCR nu/nu mouse.

Image courtesy of Michael Chiorazzo, Elizabeth Browning and Jim Delikatny, Small Animal Imaging Facility, University of Pennsylvania.

Ask for a quote today. Get your lab up and running quickly – it’s easy to use so little training will be needed for even novices. And, you can count on the reliable data for your in vivo imaging research from the Pearl Trilogy Small Animal Imaging System.

Your Commitment to Producing Reproducible Research is Critical

Sign up for LI-COR’s “Reproducibility in Science Webinar” Series. The first webinar is on March 25, 2015.

Reproducibility is becoming a highly discussed issue in all research sciences. The ability for major research findings to be independently replicated after an initial experiment is essential to building upon foundational discoveries. When experiments are not conducted thoroughly or published articles lack sufficient details for replication, we lose the ability to move ahead with accurate science. This is a major problem for researchers today.

NIH QuoteThis problem will only begin to be addressed if institutions, universities, industry, and others alike take on the responsibility of producing scientific experiments and reporting scientific methods that can be replicated at a later date. Thus, the conventions of reproducible science are paramount to the future of biomedical research findings in particular.

Several areas are being scrutinized in the discussion on biomedical reproducibility. Including:

  • Thoroughness of experimental details in journal articles
  • Review of studies submitted to journals
  • Scientific fraud
  • Utilization of highly reproducible techniques

reproducibility initiative logo smallThoroughness in research is important, because without knowing all the details of a foundational experiment future scientists are unable to efficiently build upon that research. To increase thoroughness, the Reproducibility Initiative, headed by Elizabeth Iorns, is advising full disclosure of experimental procedures in published papers. The initiative aims to identify and reward high quality, published research that can be successfully reproduced by independent validation labs. The first step in this process is pinpointing a pool of research that is true and accurate —a task The Reproducibility Initiative has begun by investigating 50 of the most impactful cancer biology studies from 2010 – 2012.

In light of the growing concern regarding scientific reproducibility, the review processes for scientific journal submissions are seeing stringent changes as well. The plans to increase the reproducibility of published papers laid out by the National Institutes of Health (NIH) at the beginning of the year are just one example. In their plan the NIH instituted a training module for enhancing the transparency of cited methods, provided a checklist for routine evaluation of grant applications, and began to urge scientific journals to revise their current review practices. Since then, high-impact journals like Nature and Science have implemented precautionary statistical checklists intended to qualify submitted research papers before publishing them in their magazines.

Unfortunately, though, there are times reported science is proved to be inaccurate, and fraudulent papers claiming breakthrough research are retracted. These retractions can severely affect scientists who have based their careers on such published inaccuracies.

ireland flag smallIn response, Ireland has taken precautions against fraudulent publication. By the end of the year The Science Foundation Ireland will be funding auditors at leading universities. The auditors will look into best practices related to research, procedures “for reporting and investigating misconduct; whether management has followed those procedures in real cases; and whether any investigations have been carried out to a satisfactory standard.” The purpose of these audits is to encourage researchers to take protocols seriously and to put standards in place that will decrease the likelihood of scientific fraud occurring.

Another area of the reproducibility discussion highlights the need for highly consistent research techniques and instrumentation. The nature of complex research and varying protocols between labs can cause inherent fluctuating results from experiment to experiment. To help combat the variability, there is a need for improved and consistent training of researchers using Western blotting and other scientific techniques in their research, just as there is a need for the instruments researchers use to be of the highest quality and to generate reproducible results. Putting more emphasis on training researchers and utilizing the highest quality instruments will help to improve the reproducibility of the studies research labs are currently conducting.

Only time will tell if the scientific community will really begin to take the issues and repercussions of reproducible science seriously. While science is shifting it is important you stay ahead of the curve and close the gaps in your research confidently. Your commitment to producing reproducible research is critical to redressing the reputation of the scientific method from beginning research stages to the published piece.

Are your findings reproducible? Read more about how reproducibility is affecting the life sciences and where the future of Western blotting may be headed.

If you’d like to learn more about reliable instrumentation, check out LI-COR Imaging Systems, which offer a digital imaging solution that ensures reproducible results. See how LI-COR can help you improve your research.

Need Visible Fluorescent Secondary Antibodies for Flow Cytometry or Microscopy?

If you are doing flow cytometry or microscopy and need dye-labeled secondary antibodies in the visible fluorescence range, we can help. LI-COR now offers IRDye® and VRDye™ dye-labeled secondary antibodies for 650nm, 549nm, and 490nm detection.

VRDye secondary antibodies are are highly cross-adsorbed – just like our IRDye secondary antibodies, making them suitable for multi-color detection.

Here is an example of immunofluorescence staining using VRDye 490 Goat anti-Rabbit Secondary Antibody.

Immunofluorescence Staining of Tubulin in HeLa Cells.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 (P/N 926-49020). Nuclei were stained with DAPI. Image acquired with Olympus IX81 microscope.

Are you ready to try IRDye Infrared Dyes and secondary conjugates or VRDye visible fluorescent secondaries on your epifluorescent microscope? Check out the recommended configurations for Olympus and Zeiss microscopes – and go image!

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

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!

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.