Is DNA Gel Documentation Important to You?

If so, do you know that the Odyssey® Fc Dual-Mode Imaging System now offers you the advantage of imaging DNA gels stained with ethidium bromide (EtBr), SYBR® Safe, and many other DNA stains using the 600 nm channel? How about that for multi-functionality?!

DNA or nucleic acid gel documentation is a common technique performed in the lab. Ethidium bromide is a common DNA stain. But, like many, if you are using SYTO® 60 as a near-infrared fluorescent DNA stain, then you can image your nucleic acid gel in the 700 nm channel of the Odyssey CLx, Odyssey Sa, OR Odyssey Fc. The detection sensitivity and lower limit of detection for SYTO 60 with any of these Odyssey imaging systems has proven to be better than with ethidium bromide detected with either a Polaroid camera or a CCD imaging system.

Don’t believe it? Check the data below, we think you may like what you see. In the figure below, DNA Gels imaged on the Odyssey Fc using Ethidium Bromide, SYBR Safe, and SYTO® 60. The Ethidium Bromide gel was also documented using Polaroid to show the comparison.
DNA Gels imaged on the Odyssey Fc using Ethidium Bromide, SYBR Safe and SYTO 60. All were imaged on the Odyssey Fc Imaging System.

Check out our technical notes on DNA gel documentation:
Imaging Nucleic Acid Gels on the Odyssey Fc Imaging System
SYTO 60 Staining of Nucleic Acids in Gels

Odyssey® References for Neuroscience Research Applications

Otis Spunkmeyer Chocolate Chip Cookie Odyssey Infrared Imaging Systems and LI-COR® reagents have been used for neuroscience research applications in many different disciplines, including neurobiology, Alzheimer’s Disease, Parkinson’s, neural tumors, and more.

This week the Society for Neuroscience is holding its annual meeting in New Orleans, LA. LI-COR is there showcasing the Odyssey CLx and the Odyssey Fc, plus the MPX™ Multiplex Blotting System, and several new products. Stop by Booth 2713 and talk to our LI-COR representatives. Fresh chocolate chip cookies will be available Monday and Tuesday at the booth during certain times – while supplies last! (follow us on Twitter – @licorbio – to find out when to stop by and get a fresh cookie snack!)
LI-COR Brain Pen

Plus pick up your free ‘brain’ pen at the LI-COR Booth (#2713).

Enjoy the conference! We wish you continued success in your research!

Here are a few journal references related to research in the Neurosciences:

Conditional Deletion of Notch1 and Notch2 Genes in Excitatory Neurons of Postnatal Forebrain Does Not Cause Neurodegeneration or Reduction of Notch mRNAs and Proteins
Jin Zheng, Hirotaka Watanabe, Mary Wines-Samuelson, Huailong Zhao, Thomas Gridley, Raphael Kopan, and Jie Shen
J. Biol. Chem., Jun 2012; 287: 20356 – 20368.

Enhancement of Rostral Ventrolateral Medulla Neuronal Nitric-Oxide Synthase–Nitric-Oxide Signaling Mediates the Central Cannabinoid Receptor 1-Evoked Pressor Response in Conscious Rats
Badr Mostafa Ibrahim and Abdel A. Abdel-Rahman
J. Pharmacol. Exp. Ther., Jun 2012; 341: 579 – 586.

Specific Serine-Proline Phosphorylation and Glycogen Synthase Kinase 3β-directed Subcellular Targeting of Stathmin 3/Sclip in Neurons
Sara Devaux, Fabienne E. Poulain, Véronique Devignot, Sylvie Lachkar, Theano Irinopoulou, and André Sobel
J. Biol. Chem., Jun 2012; 287: 22341 – 22353.

For more journal references citing the use of the Odyssey Imagers and LI-COR reagents, see our most recent Publications Lists.

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.

Select the Primary Antibodies You Use with Care for Western Blot Success

“Know Thyself – and Thy Primary Antibody!”

Okay, so you’ve done your experiments, run your sample on a gel, and transferred the proteins to a membrane. Now, you need to see if you can detect the protein, what happened to it, how much is there, etc.

After you block (remember we talked about how important the right blocker is), you will probe with a primary antibody (that is, an antibody produced to detect a specific antigen) to see your molecule of interest. Now, primary antibodies can be produced in a wide variety of species such as mouse, rabbit, goat, chicken, rat, guinea pig, human, etc. There are lots of suppliers of antibodies out there, so it is important to realize primary antibodies for the same antigen can perform very differently. It may be necessary to test multiple primary antibodies for the best performance in your Western blot system.

In the images below, you can see how different primary antibodies to the same target may react. Serial dilutions of NIH/3T3 lysate were probed with Akt monoclonal primary antibodies from three different vendors. All blots were blocked with 5% skim milk and detected with HRP-conjugated Goat Anti-Mouse and SuperSignal® West Dura chemiluminescent substrate. Western blots were imaged on the Odyssey Fc Chemi channel for 2 minutes, shown with normalized image display settings. You can see that the primary antibodies varied quite a bit. The number and intensity of bands you can detect and the amount of non-specific binding that occurs are definitely different for each one.

So, take a cue from ancient Greece and get to know your Primary Antibody by doing some testing and optimization.

The result = GREAT Western Blots!

Optimizing Chemiluminescent Western Blots – The Best Offense is a Good Blocker

Okay, it’s football season, and I thought the analogy fit. 🙂 Seriously, the right Blocking Buffer is critical to getting that great chemiluminescent Western blot.

Incubating the membrane in blocking buffer after the transfer step will result in enhanced sensitivity of your blot. Blocking buffer contains proteins that stick to the membrane, promoting specific binding of the primary antibody and minimizing non-specific interactions. Various blocking buffers are available, and it’s important to try several blockers to find the optimal solution for each antigen and antibody pair. There is not a best blocker for all conditions – so you will need to do some testing.

One very, very, very important thing to keep in mind is that the blocker used with HRP-conjugated secondary antibodies in the secondary antibody incubation step of chemiluminescent Western blotting cannot contain sodium azide.

Why?, you ask.

Well, sodium azide binds irreversibly to the HRP enzyme, inhibiting the binding of the substrate and slowing the chemiluminescent reaction. This results in less light production that may affect the appearance of less intense bands or even the entire blot. See the figures below – the blot on the left was done with blocker that did not contain azide; the Western blot on the right used Western blocking buffer with azide.

Blocking buffer with and without azide

Nota bene: Odyssey® Blocking Buffer (PBS or TBS) (which does contain sodium azide) CAN be used to block the blot and to dilute the primary antibody but not to block or dilute in the secondary antibody incubation step when using HRP-conjugated secondary antibodies.

On Another Note: Milk is a common blocking buffer; however, milk-based blockers that contain endogenous biotin and glycoproteins may result in higher background on the membrane when detecting with streptavidin. Milk may also contain active phosphatases that can de-phosphorylate phosphoproteins on the membrane.

Maximizing Sensitivity on Chemiluminescent Western Blots Technical Note

Go Digital with the Odyssey Fc Chemiluminescent and Infrared Fluorescent Imaging System or the C-DiGit® Chemiluminescent Western Blot Scanner!