Single Molecule Pulldown can be optimized with Allele’s Fluorescent Proteins

In the recent Nature paper, “Probing cellular protein complexes using single-molecule pull-down”, published May 26, 2011, researchers at the University of Illinois at Urbana-Champaign outline their findings for a new method for visualizing protein complexes through a single-molecule immunoassay which combines an antigen capturing chip and TIRF Microscopy. The SiMPull method captures protein complexes and the captured complexes are then visualized with fluorescent dyes or fluorescent protein tags. This is accomplished by using a microscope slide covered with biotinylated polyethylene glycol (PEG) and streptavidin bound to biotinylated antibodies. For single molecule visualization, multicolor labeling provides differentiation of subcomplexes and configurations.

The study includes two validation experiments, where study team members tagged their chosen complexes with YFP, in order to estimate YFP concentration after pulldown and subsequent imaging. They were then able to determine stoichiometric information in human kidney cells, from the isolated monomeric or dimeric YFPs, which exhibited the one and two-step decay responses.

Additionally, in another validation experiment, team members chose to use protein kinase A (PKA) because it’s two catalytic and two regulatory subunits separate in the presence of cAMP. This was accomplished by labeling the catalytic subunits of protein kinase A with YFP and the regulatory subunits with mCherry fluorescent protein. They then used a two-color SiMPull to pull down PKA. After pull-down they imaged the PKA in the presence of cAMP and without cAMP present. The YFP and mCherry signals fluoresced together, demonstrating that the catalytic and regulatory subunits were still attached to eachother. The YFP and mCherry signals did not correlate in the presense of cAMP, reaffirming the fact that the two subunits disassociate in the presence of cAMP.

Unlike other single-molecule pulldown techniques, the SiMPull does not require purified proteins. It also only requires about 10 cells of sample for protein pull-down and analysis, while traditional Western Blots require about 5000 cells. Moreover, This two-color SiMPull method could be further optimized yielding higher resolution overlay when used in combination with Allele’s mTFP1 and LanYFP, the brightest fluorescent proteins on the market.
The full article can be found at http://www.nature.com/nature/journal/v473/n7348/full/nature10016.html

New Product of the Week: High quality Anti-FLAG Monoclonal Antibody for detection or pulldown, ABP-MAB-DT006, $219/100ug.

Promotion of the Week: save 5% on all of our pre-packaged viruses. Our pre-packaged viruses are all titered at 10^8th or higher, and packaged in 5 tubes for your convenience. To redeem this offer email the code PREPACK to abbashussain@allelebiotech.com.

Tags: cAMP, Jain, LanYFP, mCherry, mTFP1, PKA, SiMPull, TIRF Microscopy, YFP

Allele’s pallet of the super star fluorescent proteins

“Photoblog”–just some fun pictures from our notebooks.

The brightest cyan, green fluorescent proteins, and the brightest ever FP in LanYFP!

Ain't they pretty?

These fluorescent proteins are representatives of the growing family or high quality, new generation FPs engineered to enable experiment previously deemed impossible.

Cells infected with lentivirus carrying mWasabi. Lentivirus carrying LanYFP will make most cells much more brighter than this.

The mWasabi is stimulating

The brightest green fluorescent protein with excellent photostability, carried on 10e8 TU/ml high titer lentivirus.

The LanFPs express well in bacteria.

The LanFPs express well in bacteria

Project planning is under way to test the cytotoxicity of lanFPs in different mammalian cell lines and in vivo with a focus on neurons.

The FPs fold so strongly that they fluorescence even in SDS-PAGE.

Can you see the FP bands in the SDS PAGE?

FPs in SDS PAGE–a closer look

Can you see them now?

FPs in gel cassette over UV lights

Invincible FPs

FPs in gel cassette under blue LED

Fluorescence in SDS page under blue LED

The purified FPs can be used as “real time” protein markers.

New Product of the Week 07/26/10-08/01/10: pCHAC-mWasabi-C for expressing mWasabi fusion through retroviral vectors.

Promotion of the Week 07/26/10-08/01/10: Get 3′ TAMRA & BHQ oligo mods for $45 ea & 3′ Dabcyl mod for $20 50 nmol syn scale only/while supplies last- use dbtkrm0726

Tags: Fluorescent proteins, FP, FPs, FRET, GFP, lab note, lentivirus, mCherry, mTFP1, mWasabi, photos, pictures

Brightest Ever Fluorescent Protein

LanYFP, identified from lancelet (also known as amphioxus, e.g. Branchiostoma floridae), has been found to have the following properties:

Excitation 513nm
Emission 524nm
Quantum yield 0.95
Extinction coefficient 150,000
pKa ~3.5
Salt insensitive 0-500mM NaCl

LanYFP has a brightness of 143! For comparison, the brightness of the previously known brightest FPs is 95 for tdTomato, and 34 for commonly used EGFP.

Allele already has been exclusively providing the brightest cyan FP in mTFP1 (brightness of 54); and the brightest green FP in mWasabi (brightness of 56). The confirmation of LanYFP as the brightest ever FP is a major milestone of Allele’s research and development efforts in the fluorescent protein field. We are currently monomerizing LanYFP and another lancelet protein, LanRFP. Once completed, the new proteins should definitely be the FPs of choice for in vivo imaging and FRET with unprecedented utilities.

Promotion of the week 062010-061610: Validated Rex1 Promoter Reporter Lentiviral Particles-1 Vial for $149.00 (ABP-SC-RREX2R1). Save $59 if place an order this week! http://www.allelebiotech.com/shopcart/index.php?c=200&sc=34

New product of the week, recombinant mTFP1, mWasabi, LanYFP, LanRFP, $159 for 125 ug, compare price for 100ug vs 125ug in other companies’ offers, you will know that you are getting a good deal from Allele.

Tags: brightest FP, fluorescent protein, Fluorescent proteins, FP, FP brightness, FPs, Lancelet, monomer FP, monomerization, mTFP1, mWasabi, RFP, YFP

Lentivirus-lanYFP Give Away

Promotion of the week 03-22-10 to 03-28-10: To help researchers get familiar with pre-packaged lentivirus, we offer free high titer lentivirus carrying a truly bright and fast maturing lanYFP (lancelet FP, new exclusively from Allele).  Infect virtually any mammalian cells by a single manipulation (pipeting) and watch cells turn green/yellow in about a day under microscope or on FACS.  Primarily a yellow FP, lanYFP will show brighter fluorescence than EGFP even when observed using standard GFP/FITC filter set.

New product of the week 03-22-10 to 03-28-10: anti-mTFP1/mWasabi polyclonal antibodies.  It is tailored-made for Alleleustrious mTFP1 and mWasabi, the brightest teal and green FPs.

Tags: EGFP, Fluorescent proteins, GFP, Lancelet, lentivirus, mTFP1, mWasabi, pre-validated virus, YFP

Fluorescent Protein-Based Assay Development

This blog is a preview of what is to be launched as a new Service Group. Allele Biotech is restructuring its CRO capabilities in the assay development area by combining its fast expanding fluorescent protein portfolio, viral vector and packaging expertise, as well as newly granted patents in shRNA. The focus of this post is fluorescent protein in biosensor and screening assays. A modified version will be used as the landing page for the FB-Based Assay Development Service.

Overview:

Originally cloned from the jellyfish Aequorea victoria and subsequently from many other marine organisms, fluorescent proteins (FPs) spanning the entire visual spectrum have become some of the most widely used genetically encoded tags. Unlike traditional labeling methods, FPs may be used to specifically label virtually any protein of interest in a living cell with minimal perturbation to its endogenous function. Genes encoding FPs alone or as fusions to a protein of interest may be introduced to cells by a number of different methods, including simple plasmid transfection or viral transduction. Once expressed, FPs are easily detected with standard fluorescence microscopy equipment.

Factors that should be taken into account when designing an FP-based imaging experiment include the desired wavelength(s) for detection, the pH environment of the tagged protein, the total required imaging time, and the expression level or dynamic range required for detection of promoter activity or tagged protein. Individual FPs currently available to the research community vary considerably in their photostability, pH sensitivity, and overall brightness, and so FPs must be chosen with care to maximize the likelihood of success in a particular experimental context.

FPs as fusion tags:

Use of FPs as fusion tags allows visualization of the dynamic localization of the tagged protein in living cells. For such applications, the cDNA of a protein of interest is attached in-frame to the coding sequence for the desired FP, and both are put under the control of a promoter appropriate to the experimental context (typically CMV for high-level expression, though other promoters may be desirable if overexpression of your protein of interest is suspected of producing artifacts). The most basic uses for fluorescent protein fusions include tracking of specific organelles (fusions to short organelle targeting signals) or cytoskeletal structures (fusions to actin or tubulin, for example). More advanced uses include tracking receptors or exported proteins. In most cases, it is critical that the FP used for fusion tagging be fully monomeric, as any interaction between fusion tags is likely to produce artifacts, some of which may be hard to recognize in the absence of other controls. While in most cases FP fusions do not interfere with normal protein function, whenever possible, FP fusion proteins should be validated by immunostaining the corresponding endogenous protein in non-transfected cells and verifying similar patterns of localization.

FPs as expression reporters:

FPs are highly useful as quantitative expression reporters. By driving the expression of an FP gene by a specific promoter of interest, it is possible to produce an optical readout of promoter activity. Use of the brightest possible FP ensures the best dynamic range for such an experiment. Because dynamic localization is not generally an issue for expression reporter applications, it is possible to use non-monomeric FPs for this purpose, opening up additional possibilities for multiple wavelength imaging. In order to obtain more reliable quantitative data and to correct for likely variations between individual cells in expression reporter experiments, the use of two spectrally distinct (e.g. green and red) FPs is advisable. By driving expression of one FP with a constitutive promoter and a second FP with the promoter of interest, the ratio of the two signals provides a quantitative readout of relative activity. Averaged over many cells, this technique should provide statistical power necessary for quality expression level experiments. Because FPs normally have a very slow turnover rate in mammalian cells, it may be desirable to add a degradation tag to your FP to enhance temporal resolution when measuring highly dynamic promoter activity.

New Product of the Week 03-08-10 to 03-14-10: mWasabi 2A or IRES dual expression vectors (http://www.allelebiotech.com/shopcart/index.php?c=216&sc=34) ABP-FP-W2A10, orWIRES10

Promotion of the Week 03-08-10 to 03-14-10: for a limited time on Thursday, to be announced on our Facebook page (http://www.facebook.com/pages/San-Diego-CA/Allele-Biotechnology-and-Pharmaceuticals-Inc/78331924957#!/allele.biotech?ref=profile), a strikingly low price will be honored for a commonly used lab reagent or equipment. This is the second week of the follow-us-to-the-basement promotion.

Tags: assay development, biosensor, cell based assays, chemical screening, circular permutation, fluorescent protein, FRET, GFP, mTFP1, mWasabi, promoter-FP, RNAi screening