Article Category: Applications

NEW! IRDye® Goat Anti-Mouse IgM Secondary Antibodies from LI-COR®!

IRDye Dye-labeled Goat anti-Mouse AntibodiesOur IRDye secondary antibody line is growing! We have recently added IRDye Goat anti-Mouse IgM (μ chain specific) secondaries labeled with:

  • IRDye 800CW (PN 926-32280)
  • IRDye 680RD (PN 926-68180) or
  • IRDye 680LT (PN 926-68080).

Just like all of the LI-COR IRDye secondary antibodies, these are highly cross-adsorbed secondary antibody conjugates suitable for a variety of applications (see the table below).

IRDye 800CW secondary antibodies are the antibodies of choice for a wide variety of applications in the 800 nm channel (see the list below). IRDye 800CW secondary antibodies can be used for 2-color detection when multiplexed with IRDye 680RD or IRDye 680LT secondary antibodies.

IRDye 680RD secondary antibodies are the antibodies of choice for In-Cell Western Assay and Western blot applications in the 700 nm channel. These antibodies can be used for 2-color detection when multiplexed with IRDye 800CW secondary antibodies. These antibodies are our most universal use 700 nm channel antibodies. Start using IRDye 680RD first over other 700 nm dyes. Dilution working range 1:10,000 – 1:40,000.

IRDye 680LT secondary antibodies have been proven the brightest signal for Western blot detection in the 700 nm channel and are comparable to Alexa Fluor 680 secondary antibodies. Choose IRDye 680LT secondary antibodies to get high signal and for specific uses of detection in the 700nm channel. These antibodies are not recommended when getting up and running on system. Once established near-infrared protocols are optimized with IRDye 680RD, IRDye 680LT can be used to optimize signals in the 700 channel. Dilution range 1:20,000 – 1:40,000. Note: optimization may be required with IRDye 680LT.

Application IRDye 800CW
Secondaries
IRDye 680RD
Secondaries
IRDye 680LT
Secondaries
Western Blot
In-Cell Western™ Assay Not Recommended
On-Cell Western Assay Not Recommended
Protein Array
Immunohistochemistry
Microscopy
2D Gel Detection
Tissue Section Imaging
Small Animal Imaging Not Recommended
Virus Titration Assay Not Known Not Known
FRET-based Assay Not Known Not Known


Note: Now, as of December 15, 2014, you can also get 0.1 mg sizes of all of our IRDye dye-labeled secondary antibodies. Check out our complete listing here and our new filtering tool!

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

Reprobe Fluorescent Westerns with NewBlot™ Western Blot Stripping Buffers

NewBlot Stripping Buffer IconNewBlot Western Blot Stripping Buffers are specially formulated for use with IRDye® infrared dyes (680RD, 680LT, and 800CW only) and the Odyssey® Infrared Imaging Systems to help you save time and money on recreating samples. NewBlot Stripping Buffer allows you to reuse the same blot by stripping and reprobing up to two fluorescent antibodies simultaneously.

So, you may ask, what’s so great about NewBlot Stripping Buffer?

  • Effectively removes antibodies, yet gentle enough to retain immobilized proteins
  • Strips blots at room temperature in 20 minutes or less without an unpleasant odor
  • Allows you to reuse the same blot up to 3X! (see the data below!)
  • Offers qualitative analysis after stripping

In the example below, beta tubulin and ERK2 were run on a gel and transferred to Immobilon®-FL PVDF membrane. They were probed with primary antibodies rabbit anti-beta-tubulin and mouse anti-ERK2 and then with IRDye 680 Goat anti-Rabbit (red) and IRDye 800CW Goat anti-Mouse (green), respectively. NewBlot PVDF Stripping Buffer was used to strip the blot, which was then reprobed with the fluorescent secondary antibodies. This was repeated 2 more times. As you can see from the series of images, there is very little apparent loss of signal in either channel in the third blot as compared to the original blot.
Example of using NewBlot Stripping Buffer on PVDF Membranes

NewBlot is available in two ‘flavors’: one for stripping nitrocellulose membranes and the other for stripping PVDF membranes.

Get more power out of your blot with NewBlot Western Blot Stripping Buffers!

Note: On August 25, 2014, we launched two new Western blot stripping buffers: NewBlot™ IR Stripping Buffer for infrared Western blots on either PVDF OR nitrocellulose membranes; and, WesternSure® ECL Stripping Buffer for chemiluminescent Western blot stripping and reprobing. BOTH do not require hazardous shipping charges, unlike many other Western stripping buffers.

NEW! COX IV Primary Antibody Offers Normalization for Low-Expressing Proteins

COX IV Rabbit Monoclonal Primary Antibody, PN 926-42214The COX IV primary antibody can be used for detection of COX IV by Western blot, or as a normalization antibody when performing two-color detection. Its target molecular weight is 17 kDa. Detection of this primary antibody can be achieved with IRDye® Goat Anti-Rabbit or IRDye Donkey Anti-Rabbit secondary antibodies. LI-COR® also carries beta-actin, alpha-tubulin, and beta-tubulin primary antibodies for normalization when performing quantitative Western blots or In-Cell Western Assays.

Western Blot Linearity of COX IV vs. Actin in Cell Lysate

Figure 1. Linearity comparison of COX IV rabbit monoclonal primary antibody (P/N 926-42214) to β-Actin rabbit monoclonal (P/N 926-42210). Primary antibodies were compared by Western blot and detected with IRDye 800CW Goat anti-Rabbit (P/N 926-32211). The COX IV antibody can be used as a mitochondrial loading control and a loading control for normalizing low expressing target proteins. This COX IV primary antibody remains linear with increasing concentrations of lysate, making it ideal for normalization.

COX IV Rabbit Monoclonal Antibody Pack Insert

Use IRDye® Labeled Oligonucleotides for Safer, Faster Fluorescent Gel Shift Assays

The EMSA (electrophoretic mobility shift assay) is used to study protein:DNA complexes and interactions. Protein:DNA complexes migrate more slowly than unbound linear DNA on a non-denaturing gel, causing a “shift.”

Also called “gel shift” or “gel retardation” assays, EMSA can be used to analyze sequence-specific recognition of nucleic acids by proteins.

Traditional, radioactive EMSA protocols can be easily adapted to near-infrared fluorescence EMSA detection by using IRDye end-labeled oligonucleotides and imaging with the Odyssey® CLx or Odyssey Classic Infrared Imaging System, providing a safe and sensitive alternative.

Comparison of Detection Methods for Fluorescent Gel Shift Assay

For more information on the EMSA workflow and a sample protocol for infrared fluorescent mobility shift assays, visit our website.

In-Cell Western™ Assay Webinar – Applications Review

In-Cell Western Assays - Fluorescent ImmunoassaysFor those of you that like to watch videos and listen to information, here is a great webinar on In-Cell Western Assays.

In this In-Cell Western webinar, the basics of ICW assays are covered and these applications:

For more information on these plate-based fluorescent immunoassays, go to the In-Cell Western assay application page. There are also several sample protocols and information on how to set up, optimize, and analyze ICW assays.

In-Cell Western™ Assay Application: Response of COS-7 Cells to Hydroxyurea

Application: Detecting phospho-p53 in COS cells in response to Hydroxyurea


Example of In-Cell Western Assay: Effects of Hydroxyurea on phospho-p53 on COS-7 cells

In this In-Cell Western assay application, the response of COS-7 cells to increasing doses of hydroxyurea was measured by a specific antibody (Anti-phospho-p53 from Cell Signaling Technology, P/N 9286) that detects phosphorylated-p53 (Ser16). Total ERK1 was used for normalization. The image represents a 96-well two-color In-Cell Western with the 700 and 800 nm channels detecting phosphorylated-p53 (Ser16) and total ERK1, respectively. Background wells were incubated with secondary antibody but no primary antibody. IRDye® 680RD secondary antibodies were used for detection in the 700nm channel and IRDye 800CW secondary antibodies were usd for detection in the 800nm channel.

Dose response graph of % induction of p53 phosphorylation with hydroxyurea in COS-7 cells

The graph represents the average of four sets of quantitative data, demonstrating the percent induction of phosphorylated-p53 (Ser16). Plate-based assays such as this can be imaged on the Odyssey® CLx or Odyssey Sa Infrared Imaging System.

For more uses of In-Cell Westerns Assays, visit our website.

In-Cell Western™ Assay Quality Assessment using Z’-Factor

If you have been following my posts for the past two months or so, you know we have been looking a great deal at In-Cell Western Assays (also called In-Cell ELISAs or plate-based immunofluorescent assays). On April 3, I explained what an ICW Assay is. From there we looked at examples of how In-Cell Westerns are used (studying apoptosis, for IC50 Determinations), seeding plates for In-Cell Westerns), ICW kits LI-COR® offers, and using cells in suspension, plate selection for ICW Assays, and cell lines that have been tested for use with this powerful immunocytochemical assay.

So you are hopefully ready to give this technique a try. When you do, it is important to assess the overall quality and reliability of the assay during In-Cell Western (ICW) assay optimization. The Z’-factor statistic provides a way to evaluate whether or not assay conditions (reagents, protocols, instrumentation, kinetics, and other conditions not directly related to the test compounds) are optimized. Z’-factor, introduced by Zhang et al., is a dimensionless value that represents both the variability and the dynamic range between two sets of sample control data.

Z’-factor experiments are performed on one or more ICW assay plates containing replicate wells designated for background subtraction, negative control samples, and positive control samples. Typically, negative control wells are those in which the cells receive an the appropriate treatment so as to elicit the lowest desired percent response (usually untreated cells); positive control wells are those in which the cells receive an appropriate treatment so as to elicit the maximum desired percent response; background wells are treated the same as the negative control wells, except primary antibody incubation is excluded.

Example of Z' Factor Data Plot

Here are some resources so that you can read more about the Z’-Factor, how to set up the experiments to assess it, and its importance in ensuring you have a high quality, reliable assay method:
Using the Z’-Factor Coefficient to Monitor Quality of Near-Infrared Fluorescent Cell-Based Assays
In-Cell Western Assay Quality Assessment Using Z’-Factor Data Sheet

Read more about Reproducibility and Precision in In-Cell Western Assays.

Cells in Suspension for Quantitative Cell Signaling Analysis using In-Cell Western™ Assays

In-Cell Western Assays - Fluorescent Immunoassays

So I’ve talked about how to ensure that suspension cells attach to plates, when to know you have a monolayer, and why round bottom plates are the best when doing In-Cell Western Assays with suspension cells in the 23-May-12 blog post. On 29-May-12 post, the post discussed how to wash your microplates so that you don’t lose cells plus some troubleshooting tips. I hope you found both of those posts helpful.

Here is the last post in this series on using non-adherent cells for ICW assays. These are a few additional questions you may have about using suspension cells for this powerful immunofluorescent assay.

  1. What suspension cell lines have been tested for use in In-Cell Westerns?
    Suspension cell lines tested include Jurkat, K-562, and THP-1.
  2. What pathways have been tested?
    Pathways tested include ERK activation and apoptosis using cleaved caspase-3 as a marker (Figure 1). A sample protocol can be downloaded here.

Do you have other questions? Super! Please contact us and let us know how we can help you in your research. And stop by this blog again for more technical tips and troubleshooting hints on other applications.

Anisomycin induced Apoptosis in Jurkat Cells

Figure 1. Anisomycin-induced apoptosis in Jurkat cells. The image represents a 96-well two-color In-Cell Western assay with the 700 and 800 nm channels detecting TO-PRO®-3 DNA staining and cleaved caspase-3 (Asp175), respectively. The image was scanned using the Odyssey® Sa Infrared Imaging system with scan setting of 200 μm resolution, focus offset of 3.5, and intensity of 3.5 (700 channel) and 4 (800 channel). Background (B) wells were incubated with a secondary antibody but no primary antibody. The graph represents normalized quantitative data demonstrating the increase in caspase-3 cleavage in response to anisomycin treatment for 3 hours in Jurkat cells.

Additional resources:
In-Cell Western Assays: FAQs when Using Suspension Cells
Complete Sample Protocol for PMA-Induced ERK Activation in Suspension Cell Lines
LI-COR BIO Website

More Essential Success Tips for Performing In-Cell ELISAs Using Non-Adherent Cells

Round Bottom Plates for In-Cell Western Assays

I mentioned in my post on 23-May, that the next few entries would be on more hints and tips of how to use non-adherent cells for In-Cell Western Assays – so here goes!

During my washing steps, cells are coming off the plates.

  1. Are you using the recommended round bottom 96-well plate (BD Biosciences, P/N 353077)?
    1. If no, cells will more easily detach from the flat bottom plates than the round bottom
      plates. The multi-channel pipettors will generate enough pressure when expelling liquid from the pipet to cause cell detachment when using flat bottom plates. Cells will detach even when pipetting down the sides of the wells.
    2. If yes, make sure you pipet down the sides of the wells and not directly onto the cells. If this doesn’t help, you may need to change your multi-channel pipettor because different brands of pipettors have different amount of pressure required to expel the liquid from the pipet. The recommended multi-channel pipettor is the 12-channel Finnpipette
      (Thermo Electron Corp, P/N 4610050).
  2. Are you shaking or rotating the plates at a moderate to high speed?
    1. If yes, gentler shaking/rotating is needed to prevent cells from detaching. Cells will detach. Set shaking or rotating speed to very low speed.
    2. If no, are you dumping the solutions straight from the plates? Dumping causes cells to
      detach. Either aspirate very slowly or manually pipet using the sides of the wells.

Why can’t I use the flat bottom 96-well plates?

  • LI-COR® Biosciences recommends using the round bottom 96-well plates for the reasons mentioned above.

When I scan an empty round bottom 96-well plate, I get lots of background noise.

  • The round bottom plate shows some background autofluorescence. The background fluorescence is relatively small compared to signal (about 200-fold difference depending on the intensity of the signal) and can be subtracted from the signal. It is necessary to include background wells containing cells and only the secondary antibodies in order to
    completely subtract away the background noise originating from the plate as well as from the non-specific binding of the secondary antibodies.

Here is a technical note on more FAQs on using non-adherent cells for In-Cell Western Assays. Or you can just stay tuned to my next blog post!

To your Research Success!