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

Originally published at AlleleBlog:

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.

Allele’s pallet of the super star fluorescent proteins

From AlleleBlogs
http://allelebiotech.com/blogs/2010/07/alleles-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

Expanding the Camelid Antibody Product Line

From AlleleBlog http://allelebiotech.com/blogs/

While Chromotek GFP-Trap resin has become one of the best sellers from the Allele Biotech’ Camelid Antibody product line, more products have been added that will prove to be great tools for GFP-related research.

GFP is a powerful tool to study protein localization and dynamics in living cells. However, the photo stability and the quantum efficiency of GFP are not sufficient for Super-Resolution Microscopy (e.g. 3D-SIM or STED) of fixed samples from cells expressing GFP-fusion proteins to visualize specific structures. Furthermore, many cell biological methods such as HCl treatment for BrdU-detection, the EdU-Click-iT™ treatment or heat denaturation for FISH lead to disruption of GFP signal.

Now we offer our GFP-Trap Booster for reactivation, boosting and stabilization of GFP, suitable for acquiring strong and long lasting signals from GFP-fusion proteins. It is based on a specific GFP-binding protein as in GFP-Trap but coupled to the fluorescent dye ATTO 488 (from ATTO-TEC). For information, please read the product description of this week New Product of the Week: GFP-Trap booster, ABP-CM-GBOOSTR, http://www.allelebiotech.com/shopcart/index.php?c=221&sc=158

Promotion of the week: All mTFP1 and mWasabi fusion plasmids are 30% off for this week only

Preview of future new product: a similarly high quality product, the RFP-Trap that pulls down DsRed derived proteins including mRFP1, mCherry, mOrange, mPlum but also mRuby and RFP-tagged fusion proteins.