ProbesOnline - May, 2011

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In This Issue


Gibco® Primary Human Corneal Epithelial Cells (HCECs)   Get More Physiologically Relevant Results — Gibco® Primary Human Corneal Epithelial Cells (HCECs)
ABfinity™ Recombinant Rabbit Monoclonal Antibodies   Study c-Met Activation With New Recombinant Antibodies — ABfinity™ Rabbit Monoclonal Antibodies
Tau [pT231] Human ELISA Kit   Measure Phosphorylation of Tau Proteins — Tau [pT231] Human ELISA Kit


Click-iT® Glycoprotein Profiling Reagents   Discovering O-GlcNAc Sites on Proteins — Click-iT® Glycoprotein Profiling Reagents



Molecular Probes® Social Media   Molecular Probes® Photostream!
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Get More Physiologically Relevant Results — Gibco® Primary Human Corneal Epithelial Cells (HCECs)

what they are
Human Corneal Epithelial Cells (HCECs) from Gibco® are normal human cells isolated from the progenitor-rich limbal region of the eye, and are cryopreserved at the end of the secondary culture. HCECs are ideal for research on corneal biology, including inflammation and wound healing, investigating the effects of chemicals and components in consumer products, and other studies of ocular function.

what they offer

  • Guaranteed to be ≥70% viable upon thawing and to contain ≥500,000 viable cells/vial
  • Guaranteed to reach at least 12 population doublings after thawing when using Keratinoctye-SFM
  • Stain positive for corneal epithelial markers cytokeratin 15 and p63 alpha

how they work
Cryopreserved HCECs are optimal for investigating the cellular, molecular, and biochemical processes that occur in the cornea and have been optimized for use with Gibco® Keratinocyte Serum-Free Medium. HCECs closely mimic the in vivo state and enable generation of physiologically relevant data. Limbal tissue is known to be enriched for corneal epithelial progenitor cells. Our robust isolation and expansion process yields large single-donor lots, to help ensure availability of consistent product.

Study c-Met Activation With New Recombinant Antibodies — ABfinity™ Recombinant Rabbit Monoclonal Antibodies

what it is
The c-Met receptor is a potential therapeutic target in some cancer models. c-Met is activated by phosphorylation at tyrosine residues, and can be activated by epidermal growth factor receptor (EGFR) without production of hepatocyte growth factor (HGF), inducing signaling cascades through a ligand-independent event.

what it offer

  • ABfinity™ antibodies—recombinant antibodies enable consistent results, time after time
  • New antibodies released every month


how it works
ABfinity™ recombinant rabbit monoclonal antibodies help ensure consistent antibody performance lot after lot, so you don’t have to revalidate dilutions for your experiments when you order more. c-Met ABfinity™ Recombinant Rabbit Monoclonal Antibodies are available for total c-Met and c-Met phosphorylated at tyrosine 1230, and are validated in applications ranging from western blotting to immunofluorescence. ABfinity™ Recombinant Rabbit Monoclonal Antibodies are also available for EGFR.

Immunocytochemistry of A549 cells labeled with rabbit anti-c-Met antibody

Immunocytochemistry of A549 cells labeled with rabbit anti–c-Met antibody.

Alexa Fluor® 488 goat anti–rabbit IgG at 1:1,000 dilution was used as the secondary antibody. Actin was stained with Alexa Fluor® 568 phalloidin; nuclei were stained with Hoechst stain. Left: Hoechst only. Middle: c-Met signal only. Right: composite image.

Measure Phosphorylation of Tau Proteins — Tau [pT231] Human ELISA Kit

what it is
The Invitrogen™ Tau [pT231] Human ELISA Kit is designed to detect and quantify the level of threonine-231 phosphorylation in tau proteins from human cell lysates and cerebrospinal fluid (CSF). This validated and complete phosphoELISA™ kit contains all the buffers and reagents you need to quickly and easily perform the assay and obtain reliable results.

what it offers

  • Validated on SH-SY5Y cell lysates and CSF
  • Rapid 4-hour protocol
  • Precoated removable 8-well strips

how it works

The Invitrogen™ Tau [pT231] Human ELISA Kit is a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA).

A monoclonal antibody specific for tau (regardless of phosphorylation state) has been coated onto the wells of the microtiter strips provided. Samples are pipetted into these wells. During the first incubation, the tau antigen binds to the immobilized (capture) antibody. After washing, a rabbit antibody specific for tau [pT231] phosphorylation is added to the wells. During the second incubation, this antibody serves as a detection antibody by binding to the immobilized tau protein captured during the first incubation. After removal of excess detection antibody, a horseradish peroxidase–labeled anti-rabbit IgG (anti-rabbit IgG HRP) is added. This binds to the detection antibody to complete the four-member sandwich. After washing, a substrate solution is added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of tau [pT231] present.

Product Quantity Cat. No.
Tau [pT231] Human ELISA Kit 96 tests KHB8051


Discovering O-GlcNAc Sites on Histones — Click-iT® Glycoprotein Profiling Reagents

Many proteins can be regulated by the attachment of O-linked β-N-acetylglucosamine residues ( O-GlcNAc) to serine and threonine amino acids, altering their functionality in a manner similar to phosphorylation. In a recent publication, Sakabe et al. [1] utilized a combination of click chemistry and mass spectroscopy to determine the O-GlcNAc modification sites on histones.

In this study, the researchers isolated and digested the histones, yielding peptides. These peptides were then treated with phosphatase, leaving O-GlcNAc modifications intact. Then they used a product similar to the Click-iT® O-GlcNAc Enzymatic Labeling System, wherein β-1,4-galactosyltransferase (Gal-T1) transfers azido-modified galactose (GalNAz) from UDP-GalNAz to O-GlcNAc residues on target proteins in a highly specific reaction. For detection, the researchers used a product similar to the Click-iT® Biotin Protein Analysis Detection Kit followed by mass spectrometry to detect the peptides and thus the exact O-GlcNAc modification sites. 

This study illustrates the specificity and precision of click chemistry used to study the elusive sites of O-GlcNAc  modifications. The click reaction is highly specific and the reaction product contains an irreversible covalent bond, making the technique ideal for accurate downstream identification with mass spectrometry.

1. Sakabe K, Wang Z, Hart GW (2010) Beta-N-acetylglucosamine (O-GlcNAc) is part of the histone code. Proc Natl Acad Sci U S A 107(46):19915–19920.

  Click-iT® Glycoprotein Detection

Click-iT® Glycoprotein Detection.
(A) Enzymatic labeling of an O-GlcNAc–modified protein with UDP-GalNAz and Gal-T1. (B) Click-iT® azide/alkyne reaction.


Anti-GFP and Anti-RFP Antibodies — Any Platform, Any Workflow

  • Sensitive and specific detection— Alexa Fluor® dye–conjugated antibodies offer optimal specificity and low background
  • Ready for any platform—choose from a variety of hosts, isotypes, and applications
Fluorescent proteins are highly versatile biological markers for monitoring physiological processes, visualizing protein localization, detecting protein expression, and purifying proteins. To complement these powerful tools, we offer anti-GFP and anti-RFP antibodies and antibody conjugates for a wide variety of applications, including imaging, western blotting, immunoprecipitation, and flow cytometry.

All six of our anti-GFP antibodies are suited for detection of native GFP, GFP variants, and most GFP fusion proteins. The affinity-purified anti-RFP antibody is used to detect native TagRFP and most fusion proteins derived from Entacmaea quadricolor. The RFP proteins derived from E. quadricolor have 2.8 times the quantum yield of mCherry, and more importantly, they remain as monomers, making it more likely that the fusion protein will function properly in the cell [1].
Anti-GFP ABfinity™ Recombinant Rabbit Monoclonal Antibody used for immunocytochemistry   Anti-GFP ABfinity™ Recombinant Rabbit Monoclonal Antibody used for immunocytochemistry. Left: U2OS cells expressing CellLight® ER-GFP were incubated with the anti-GFP ABfinity™ Recombinant Rabbit Monoclonal Antibody. Center: Cells were formaldehyde-fixed, permeabilized, and blocked in 1% BSA, then incubated with primary antibody at 1 μg/mL, followed by Alexa Fluor® 647 goat anti–rabbit IgG conjugate. Right: The merged yellow signal indicates colocalization of GFP fluorescence and the detection antibody. Nuclei were stained with Hoechst 33342.
  1. Merzlyak EM, Goedhart J, Shcherbo D et al. (2007) Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat Methods 4(7):555–557.


The View


Imaging the Golgi Complex and Tubulin in a Rat Kidney Epithelial Cell.
Cells were labeled with mouse anti-giantin and rabbit anti-tubulin primary antibodies, which were detected with Alexa Fluor® 488 donkey anti–rabbit IgG (tubulin, green) and Texas Red® goat anti–mouse IgG (Golgi, red). The nucleus was stained with Hoechst 33342. Image contributed by Michael W. Davidson, Florida State University.


Precisely Delivered Nanomechanical Forces Induce Blebbing in Undifferentiated Mouse Embryonic Stem Cells

Hemsley AL, Hernandez D, Mason C et al. (2011) Cell Health and Cytoskeleton 3:23–34.

Recent studies have revealed that embryonic stem cell (ESC) proliferation and differentiation can be influenced by exogenous mechanical forces. To further investigate the effect of these forces on mouse ESCs (mESCs), Hemsley et al. combined atomic force microscopy with laser scanning confocal microscopy to characterize morphological and biochemical responses of mESCs to precisely delivered nanomechanical forces. The researchers identified two morphologically distinct subpopulations of mESCs: round and flattened. Using phalloidin conjugated to Alexa Fluor® 546 or Alexa Fluor® 488, immunofluorescence imaging of the cytoskeleton revealed that round cells, but not flattened cells, exhibited blebbing in response to mechanically induced forces. Flattened cells were characterized by a more highly developed cytoskeleton and a stronger mechanical link between the plasma membrane and cytoskeleton. These results suggest that mechanosensitivity of ESCs at the earliest stages of differentiation may play an important role in ESC biology.

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