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RIP – 'ChIP' off the old block

Chromatin regulation and control has traditionally been a key area of focus for gene expression research. More recently, the dynamics of RNA regulation have gained interest as the missing link between transcriptional regulation and protein expression.

RNA-binding proteins (RBPs) play a crucial role in regulation of gene expression by binding to RNA through their RNA recognition motif (RRM) or RNA-binding domain (RBD), resulting in the formation of ribonucleoprotein (RNP) complexes. The binding of RBPs to target RNA affects the activity, stability, localization, and function of not only the RNA species but also associated proteins. RNA-protein interactions coupled with epitranscriptomic regulation by post-transcriptional modification of RNAs, post-translational modification of RBPs, and RNA remodeling activities of RNA helicases dynamically regulate functions of RNPs.

Several methods have emerged in recent years to map RBP-RNA interactions, allowing researchers to characterize the cellular and molecular functions of RBPs. RIP - the RNA equivalent of ChIP is one such method employed to identify target RNA associated with specific RBPs. RNPs are first immunoprecipitated using a specific antibody to the target RBP. The bound RNA is then purified, followed by quantitative RT-PCR for selected RNAs or RIP-seq for transcriptome wide identification of associated RNA species that include mRNA, non-coding RNA, rRNA, and tRNAs. RIP-CLIP (crosslinking and immunoprecipitation), and PAR (photoactivatable ribonucleoside-enhanced)-CLIP are powerful tools to accelerate research.

Critical to the success of RIP is the availability of antibodies that are validated for these applications. Here we demonstrate the use of RIP for validating antibodies against 3 members of the insulin-like growth factor-2 mRNA-binding proteins (IGF2BP) family of RNA-binding proteins.

The IGF2BPs (IGF2BP1, IGF2BP2, IGF2BP3) bind to their target RNAs such as IGF2, MYC, and ACTB mRNA in either the cytoplasm or at the site of transcription in the nucleus and regulate their functions. Antibody specificity was established by demonstrating binding of IGF2BPs to 3 different known target mRNAs – IGF2, MYC and ACTB, as well as lack of significant binding to the negative control 18S rRNA.

Detection of binding of endogenous IGF2BP1 proteins
Fig. 1a
Detection of binding of endogenous IGF2BP2 proteins
Fig. 1b
Detection of binding of endogenous IGF2BP3 proteins
Fig. 1c

Detection of binding of endogenous IGF2BP proteins (IGF2BP1, IGF2BP2, IGF2BP3) to specific mRNA using Anti-IGF2BP Antibodies: RNA Immunoprecipitation (RIP) was performed using ABfinity Anti-IGF2BP1 (Fig. 1a), Anti-IGF2BP2 (Fig. 1b), Anti-IGF2BP3 (Fig. 1c) Recombinant rabbit oligoclonal antibodies (Product # 712138, 712137, 712139, 4 µg) on clarified whole cell lysate from 2 million Hep G2 cells. Normal rabbit IgG was used as a negative IP control. Immunoprecipitated RNA was purified by RiboPure RNA Purification Kit (Product # AM1924) and analyzed by RT-PCR using the Power SYBR Green RNA-to-CT 1-Step Kit (Product # 4389986) with primer pairs over IGF2, MYC, ACTB mRNA (positive) and 18s rRNA (negative). Data is presented as a fraction of immunoprecipitated RNA (%IP) normalized to the total amount of RNA used for immunoprecipitation.


A ChIPper western blot

Chromatin immunoprecipitation (ChIP) is routinely used to assess DNA-protein interactions and over time has transformed our ability to study epigenetic marks and regulatory sequences in the genome. It is used to determine the region of the DNA to which the protein of interest binds, allowing researchers to decode complex gene expression regulatory networks and DNA transactions, including DNA replication, recombination, and repair.

Choosing an antibody for ChIP depends largely on the target protein and the availability of antibodies against that protein. This process is fairly simple when the protein of interest is well-studied in different cell types and well-characterized antibodies are commercially available. Full characterization of an antibody, however, is not a determinant of its performance in cross-linked ChIP. Cross-linking may potentially modify or mask epitopes leading to the loss of specific epitopes or the formation of new ones. In cases where the protein of interest is not well-studied, and the market lacks ChIP-grade antibodies, you can check whether your antibody will work in ChIP by performing ChIP followed by western blot (ChIP-WB). This technique also comes in handy when the genomic sequences to which the protein binds are unclear.

ChIP-WB involves running the immunoprecipitated proteins obtained from a chromatin lysate prepared from cross-linked cells on SDS-PAGE, followed by blotting and probing with either the same or a different antibody against the protein of interest. Presence of immunoreactive bands of expected molecular weights only in the immunoprecipitated and input chromatin lanes confirms successful immunoprecipitation, and indicates that the protein of interest is being pulled down.

Here we demonstrate the use of for the validation of Lamin A/C antibodies. Lamins are structural components of the nuclear membrane, and play a vital role in various cellular functions such as DNA replication and cell cycle regulation.

Chromatin immunoprecipitation-western blot (ChIP-WB)

Chromatin immunoprecipitation-western blot (ChIP-WB) of endogenous Lamin A/C proteins using Lamin A/C antibody: ChIP was performed using Lamin A/C Mouse Monoclonal Antibody (Product # MA1-06102, 10 ug) (lane 2) on sheared chromatin from 4 million formaldehyde fixed HeLa cells. Normal rabbit IgG (lane 1) was used as negative IP control. Western blot analysis was performed on immunoprecipitated proteins using Lamin A/C Monoclonal Antibody (Product # MA1-5820, 1:200 dilution). Bands at 74 kDa and 65 kDa correspond to Lamin A and Lamin C respectively.

NEW PRODUCT RELEASES

New Alexa Fluor Plus Secondary antibodies

More species, new Alexa Fluor Plus 594 dye conjugates now available. The Alexa Fluor Plus antibodies are tailored to to detect low abundant proteins and to perform well in challenging experimental systems. These antibodies have superior signal-to-noise ratios, allowing the best possible results.

What is new:

  • One new dye: Alexa Fluor Plus 594
  • Six new species combinations across all Alexa Fluor Plus conjugates

The Alexa Fluor Plus portfolio now has 36 antibodies that offer:

  • Higher signal-to-noise ratio allowing detection of low abundant targets
  • High cross-adsorbtion to minimize cross-reactivity and low background
  • Improved sensitivity and range of linear detection to provide more detail
  • Multiplexing capabilities in ICC and fluorescent WB
  • Conjugates chosen for best signal-to-noise performance using high throughput screening, multiple rounds of validation* and confirmation for performance in multiplex workflows

Alexa Fluor Plus conjugated secondary antibodies are available only in the Invitrogen portfolio at Thermo Fisher Scientific.

Western blot analysis of phosphorylation of EGFR and its downstream target – MEK1 was performed by loading 30 µg of A-431 Control (lane1), A-431 treated with EGF (200 ng/ml for 10 minutes) (Lane 2), A-431 CAS9 (Lane 3), A-431 CAS9 treated with EGF (200 ng/ml for 10 minutes) (Lane 4), A-431 EGFR KO (Lane 5), A-431 EGFR KO treated with EGF (200 ng/ml for 10 minutes) (Lane 6) whole cell extracts using Novex NuPAGE 10 % Bis-Tris gel, XCell SureLock Electrophoresis System (EI0002), Novex Sharp Pre-Stained Protein Standard (LC5800), and iBlot Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature. Phospho EGFR was detected at ~160 kDa using Rabbit polyclonal Antibody (PA5-17848), EGFR was detected at ~160 kDa using Mouse Monoclonal Antibody (MA5-13343), Phospho MEK1 was detected at ~ 45 kDa using Rabbit monoclonal Antibody (702581) ABfinity Rabbit Monoclonal Antibody, MEK1 was detected at ~45 kDa using Mouse Monoclonal Antibody (MA5-15093), GAPDH was detected at ~37kDa using Goat Polyclonal antibody (PA1-9046) at 1:1000 dilution in 5% skim milk at 4°C overnight on a rocking platform. Alexa Fluor Plus Donkey anti Mouse 680, 800 conjugates; Alexa Fluor Plus Donkey anti Rabbit 680,800 conjugates and Alexa Fluor Plus Donkey anti Goat 488 and 555 conjugated secondary antibodies were used to detect the target proteins. EGF induced phosphorylation of EGFR and MEK1 was observed in control cell line (lane 1-4) and not in the EGFR knockout cell lines (lane 5,6).

Alexa Fluor Plus packaging

These new antibodies will be shipped in the same Alexa Fluor Plus packaging that saves time and space: clear vials with flagless labels and color coded caps corresponding to the respective dye conjugate.

Learn more about the Alexa Fluor Plus conjugates and the species combinations now available at thermofisher.com/alexafluorplus

*The use or any variation of the word "validation" refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical or diagnostic uses.


New Invitrogen antibodies for flow cytometry

Thermo Fisher Scientific now offers >13,000 Invitrogen antibodies for flow cytometry. With product releases every month, you never know what you might find! Check our search experience that:

  • Helps you quickly identify the most referenced clone
  • Data images and captions featured directly from the literature
  • Allows you to click directly through to the articles which have used the antibody that is of interest to you

Thermo Fisher Scientific now offers over 640 eBioscience Super Bright antibody conjugates for the violet laser! Additionally, check out these recently released clones:

Search now for your flow cytometry at thermofisher.com/flowantibodies


POU-er tools for your development research

Invitrogen launches recombinant ABfinity antibodies against key "POU" transcription factors known to be involved in development and cancer.

The POU2AF1 antibody (Cat. No. 703325) has been tested in western blot and ChIP assays. It, specifically picks up a signal in B cell lines (Raji, Ramos, Daudi) where it is known to be enriched, but not in T cell lines (Jurkat, MOLT4) which are negative for this protein.



Testing of ABfinity Anti-POU2AF1 antibody (Cat. No. 703325) for western blot (secondary antibody Cat. No. A27036) (A) and ChIP assay (B).

The POU3F2 antibody (Cat. No. 711869) recognizes POU3F2 in both immunofluorescence (IF) and western blot. A strong signal is picked up in immunofluorescence (IF) in neural stem cells which abundantly express POU3F2 . The specificity of this antibody is shown by the reduction in western blot signal in siRNA treated A375 cells, a melanoma cell line.



Testing of ABfinity Anti-POU3F2 Antibody (Cat. No. 711869) for immunofluorescence IF (secondary antibody Cat. No. A27034; green represents POU3F2, red represents F-Actin, and blue represents nuclei) (A) and siRNA mediated knockdown in western blot (secondary antibody Cat. No. A27036) (B).

Use our search tool to find these antibodies and other antibodies you need at thermofisher.com/antibodies

Easily monitor all phases of protein expression and purification in real time

Pro-Detect Rapid Assays
ProDetect assays
Easy-to-read bars allow you to determine if your methods are working correctly.

New Thermo Scientific ProDetect assays detect expressed tagged proteins enabling you to verify protein expression in just 15 minutes and at earlier points along the workflow, helping to prevent unnecessary loss of time and cost due to failed processes.

  • Fast and easy—obtain visual qualitative results in 10–15 min, without the need for special equipment
  • Sensitive—detect proteins or antibodies in cell culture media, lysate, or purified protein down to the ng/mL level
  • Reliable—results are similar to other antibody immunoassays, including western blot and ELISA

A wide range of targets are available:

Pro-Detect Rapid Assay Pro-Detect Rapid Competitive Assay
Target Target
DYKDDDK-His His
SUMO DYKDDDK
GST HA
MBP Streptag II
Human Fc AVI
Rabbit Fc Myc
Mouse Fc V5

Learn more at thermofisher.com/pro-detect


Tubulin Tracker Deep Red reagent

The Tubulin Tracker Deep Red reagent (docetaxel conjugate) is used for live-cell tubulin staining. This adds to our existing Tubulin Tracker Green reagent (paclitaxel conjugate) to broaden the spectra characteristics of our product offering.

Taxol/paclitaxel and taxotere/docetaxel conjugates are used for endpoint assays of cytoskeletal behavior because they can pass through live-cell plasma membranes to specifically stain polymerized tubulin. This targeted staining is useful for applications such as cell context, cell division, or neuronal studies.

Tubulin Tracker Deep Red (T34076: 300 slides, T34077: 60 slides) Ex/Em: 652/669 nm
Tubulin Tracker Green (T34075: 300 slides), T34078: 60 slides) Ex/Em: 494/522 nm

U2OS cells multiplex labeling. U2OS cells labeled using NucBlue Live ReadyProbes Reagent (R37605), CellLight Actin-GFP (C10582), CellLight Mitochondria-RFP (C10601), and Tubulin Tracker Deep Red (T34076) show superb multiplexing capability and staining specificity. Cells were imaged in Gibco HBSS buffer (14025134) containing calcium and magnesium, supplemented with 1X Probenecid solution (P36400). Images were generated using an EVOS FL Auto 2 Imaging System (AMAFD2000) with an Olympus 60X Apochromat oil objective (AMEP4694) using DAPI (AMEP4650), GFP (AMEP4651), RFP (AMEP4652), and Cy5 (AMEP4656) EVOS light cubes.

RESOURCES AND GUIDES

Just launched: complete the free Cancer Spheroid and Organoid eLearning Course!

Microscopic images of a single spheroid and of a brain organoid

The terms spheroid and organoid both refer to culture models with three-dimensional (3D) conformation that have a specific organization and architecture that cannot be achieved in 2D monolayer cell culture. This course covers the history and basic information about the applications and limitations of 3D culture models—including spheroids and organoids—that model organ physiology and disease. Throughout the course, you will have an opportunity to evaluate your learning experience using a series of interactive knowledge checks. In addition to these newly released modules, view our entire library of informative eLearning courses.

To access the Cancer Spheroid and Organoid Course, visit, thermofisher.com/elearningcancerspheroid

Book mark each of our complementary eLearning courses.

eLearning Courses

Our animated, narrated courses provide succinct, contextual information about applications that are indispensable for understanding topics related to protein and cell analysis workflows and include these topics:

  • The four pillars of Invitrogen antibody specificity validation*:
    • Pillar 1- Immunoprecipitation mass spectrometry
    • Pillar 2-Genetic modification
    • Pillar 3-Independent antibody verification
    • Pillar 4-Biological verification
  • Protein sample preparation and purification
  • T cell subset phenotypic analysis

*The use or any variation of the word "validation" refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product, or products were validated for clinical or diagnostic uses.

To access all of the eLearning modules, visit, thermofisher.com/elearningcourses


Find flow cytometry antibodies faster

Panel Builder with Primary Conj

The Invitrogen Flow Cytometry Panel Builder is a web-based tool that can help novice and expert flow cytometerists build their flow cytometry panels.

For beginners—this tool:

  • Removes the stress of pairing markers and fluorophores
  • Makes pairing markers and fluorophores quick and simple using our highly visual format

For experts—enter the panel you’ve already designed into this tool to:

  • Review the combined spectral signals of the panel with the integrated SpectraViewer.
  • Check the fluorophore spillover information
  • Quickly and easily see what products are available for your panel

We offer over 13,000 antibodies for flow cytometry, watch the video on how to use the tool and try the Flow Cytometry Panel Builder at thermofisher.com/flowpanel


Bioconjugation technical handbook

Bioconjugation technical handbook

Now available for download, the Bioconjugation technical handbook provides helpful information and resources for the crosslinking, immobilization, modification, biotinylation, and fluorescent labeling of proteins and peptides. This handbook describes the chemical reactivity, molecular properties and applications of bioconjugation reagents, as well as provides detailed information on chemical structures, spacer arm length, and more. In addition, a glossary, references and information on digital selection tools are provided. Use this guide to find the right bioconjugation reagent for your application.

Fill a short form to download your free copy today at thermofisher.com/bioconjugationhandbook


On-demand virtual event: 3rd annual 7 Steps of Protein Virtual Event

7 Steps of Protein Virtual Event

No worries if you missed the live event on September 18. The 3rd annual 7 Steps of Protein Virtual Event is now available on-demand.

This virtual event offers both broad and in-depth content, designed to give you the information and insights you need to empower your protein research every step of the way. You will receive firsthand knowledge of the latest technologies, as well as tips and tricks for the seven protein research application areas.

  • Watch valuable webinars and poster presentations—by scientists, for scientists!—to gain insights from industry experts on how to optimize your protein biology results
  • Access a wide selection resources, such as technical handbooks, white papers, how-to-use product videos, and product selection guides
  • Receive free Continuing Education (CE) credits for the presentations you attend

Register for on-demand at thermofisher.com/7steps

Speakers

Paul Haney, PhD

Senior Product Manager, Protein and Cell Analysis, Thermo Fisher Scientific
Webinar: Light Up Your Western Blots – Fluorescent Western Blotting Tips, Tricks and More

Krishna Vattem, PhD

Senior R&D Scientist, Protein and Cell Analysis, Thermo Fisher Scientific
Poster presentation: Pro-Detect Rapid Assay Lateral Flow Kits for Detecting Protein Tags and Antibody Isotyping

David Bourdon, PhD

Senior R&D Manager, Antibodies and Immunoassays, Thermo Fisher Scientific
Webinar: ProQuantum High-Sensitivity Immunoassays Offer Minimum Sample Consumption with Maximum Performance

Other presentations and webinars cover topics ranging from detection of multiple autoimmune diseases to analysis of signaling pathways like AKT/mTOR. To see the full list of presentations and webinars, visit thermofisher.com/7steps


Flow cytometry learning center–New subtopics now live

Flow cytometry learning center

Announcing the launch of the Flow cytometry learning center subtopics area on thermofisher.com. Now you can find educational assets by application.

The subtopics cover:

In addition, we have launched a new collection of guided learning resources within the Flow Cytometry Resource Library where you can find eLearning courses as well Molecular Probes School of Fluorescence modules.

Be sure to check these new additions to the Flow Cytometry learning center


BioProbes 78-Journal of Cell Biology Applications is now available

BioProbes 78 cover

This issue features new products for imaging cell structure, articles on choosing the right immunoassay format, 2D cell culture optimization, flow cytometer instrument optimization, and more.

Read the new BioProbes issue online now


Molecular Probes School of Fluorescence

Do you need to brush up on your knowledge about the basic fluorescence principles? Are you new to imaging and want to better understand how your microscope works and how to get the best images? Does the flow cytometer working principle confound you?

If you said yes to any of these question, don't worry we have got you covered. The Molecular Probes School of Fluorescence offers 3 different modules that cover Fluorescence Basics, Imaging Basics and Flow Cytometry Basics. The content is free, with no registration required. These guided learning courses are html-based and allow you to learn at your own pace. Get started today!


A primer to fluorescent western blotting

Western blotting is an important and foundational technique in life science research. While most life science researchers use chemiluminescent substrates in the detection step of western blotting, more scientists are moving towards fluorescence-based detection strategies. Fluorescence-based western blotting enables scientists to multiplex – visualize multiple proteins on the same blot, at the same time.

With advanced digital imaging instruments such as Invitrogen iBright FL1000 Imaging System, combined with highly sensitive, low-background fluorescent conjugated antibody offerings, such as the Invitrogen Alexa Flour Plus antibodies, scientists have the necessary toolsets to fully take advantage of the fluorescent western blotting technique.

Our application note, Fluorescent Western Blotting – an Introduction for New Users, includes a detailed fluorescent western blotting protocol, tips & tricks for optimization, and troubleshooting.

Download our fluorescent western blotting application note

NEWS AND VIEWS

Not for resale. Super Bright Polymer Dyes are sold under license from Becton, Dickinson and Company.