Histones are subjected to a remarkable number of post translational modifications (PTMs) that have crucial effects on gene expression, DNA replication, repair and recombination. Histone PTM antibodies have to recognize a single modification in a highly modified histone tail where neighboring modifications may be impacting the antibody’s ability to recognize its target. In choosing a histone PTM antibody, a two pronged approach is utilized where the antibody is first tested for its specificity in recognizing only the intended modification, followed by testing its performance in relevant applications like western blotting, immunofluorescence and ChIP. Peptide arrays are an excellent predictor for antibody specificity because they contain 384 peptides from the N terminal tails of histones covering 59 different PTMs in a variety of combinations. To verify specificity of our histone PTM antibodies, we undertook a systematic analysis of the antibodies in our portfolio and compared them to highly cited competitor antibodies for those specific PTMs, using peptide arrays.

Eighteen Invitrogen histone PTM antibodies were compared with competitor antibodies and the results are summarized in the table below. Eleven of these were more specific than the benchmarked antibodies and five were equally specific. The specificity was based on a greater than two fold difference between the specificity factors for binding at the target site than those for the best non-target site.

Histones undergo post translational modifications (PTMs) that serve as epigenetic signatures for gene expression. Chromatin immunoprecipitation (ChIP) is a common application to examine these PTMs at individual loci and globally to guide our understanding of many genomic transactions. Researchers are increasingly concerned about the specificity of currently available histone PTM antibodies, specifically for ChIP applications. Peptide arrays provide valuable information regarding specificity for a a large number of modifications and in the context of neighboring modified residues. However, screening by peptide arrays is in a denaturing protein context; whereas ChIP requires an antibody to recognize the modification in its biological context. A newly developed method (SNAP-ChIP: Sample Normalization and Antibody Profiling) can be employed to rigorously test antibody specificity for ChIP applications. Spiking-in a panel of recombinant semi-synthetic modified nucleosomes allows one to determine if an antibody is enriching the target of interest compared to the other histone PTMs (i.e. off-target) in the panel. The panel includes unmodified H3, H4 and mono, di and tri-methyl H3K4, H3K9, H3K27, H3K36 and H4K20. Shown below is an example of SNAP-ChIP for H3K4me1 (Cat. No. 710795). SNAP-ChIP provides a unique validation approach and represents the strongest evidence to date for the performance and specificity of histone PTM antibodies in ChIP. Our SNAP-ChIP validated antibody portfolio provides researchers the confidence in ChIP qPCR and ChIP-seq data to associate histone modifications with biological effects.

SNAP-ChIP provides a unique validation approach. SNAP-ChIP K-MetStat (EpiCypher, 19-1001) was performed to analyze the histone specificity of H3K4me1 (Cat. No. 710795) in ChIP. This ChIP assay spikes in a panel of post-translationally modified, barcoded, semi-synthetic nucleosomes during the normal ChIP workflow to determine how much of each PTM is immunoprecipitated (for more information see reference https://www.ncbi.nlm.nih.gov/pmc/PMC4458216). Cat. No. 710795 was tested in native ChIP with 3 µg chromatin from HEK-293 cells. Specificity (left Y-axis; all bars mean ± SEM from six independent ChIP experiments) was determined by quantitative real-time PCR (qPCR) to each modified nucleosome in the SNAP-ChIP K-MetStat panel (X-axis). Black bar represents antibody efficiency (right Y-axis; log scale) and indicates percentage of the barcoded H3K4me1 nucleosome target immunoprecipitated relative to Input.

 Find out more about ChIP standardization and antibody development

Chromatin immunoprecipitation (ChIP) is extensively used to assess and understand protein-DNA interactions. To ascertain that the antibody being used is only pulling down the targeted protein, one can examine regions of the genome that are known to be enriched and known to be depleted of the protein being ChIPed. The profile of histone PTMs, which are essential for maintaining genomic stability and regulation of gene expression, varies not only between active or silent genomic loci, but also across a single gene. Use of a single gene locus does not provide a complete picture of the pattern of the histone modification, which can lead to false positive or false negative ChIP results. However, when the expected pattern of pull down of chromatin at multiple loci across a gene is observed, it is indicative of antibody specificity to its intended histone modification.

For example, tri-methylation of histone H3 at lysine 36 (H3K36me3) is associated with actively transcribed genes and enriched specifically towards the 3′ end of active genes. For two different antibodies against this modification (Product # MA5-24687; = Product # PA5-17109; see below), we examined pulldown efficiencies at four different loci (upstream, promoter, gene body, and downstream regions) of two genes, beta-Actin (ACTB) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ChIP with both antibodies showed enrichment for the gene body and downstream regions of GAPDH, but not upstream or promoter regions, consistent with H3K36me3 being associated with the 3’ end of active genes. Similarly for ACTB, we observe higher fold enrichment at the gene body and downstream region compared to the upstream or promoter regions. Both antibodies show little to no enrichment of H3K36me3 at transcriptionally silent negative controls MYOD and SAT2 satellite repeats. This provides a degree of confidence that these antibodies are indeed binding to H3K36me3.

Zenon labeling technology provides a reliable method for producing fluorescently labeled antibodies with pHrodo iFL green or red labels in 10 minutes. The Zenon pHrodo iFL labeling reagents can be complexed with human or mouse IgG to provide rapid labeling for fast, scalable screening of antibody internalization for many antibody samples. pHrodo iFL labeled antibodies are essentially non fluorescent until they are internalized into acidic compartments inside the cell.

Zenon fragments are specifically designed to target and bind to the Fc portion of a human or mouse IgG antibody away from the antigen biding region. The Zenon labeling method offers unique flexibility for screening potential antibody drug conjugates providing the flexibility to look at many antibodies or varying antibody concentrations. Zenon technology can be used to label as little as1 µg of antibody, and unlike traditional labeling methods that use amine- or thiol-reactive labels, the Zenon labeling procedure is compatible with BSA and other stabilizing proteins.

We have released 200+ new flow cytometry antibody conjugates to provide you more options when designing your flow cytometry panels. These new products include:

• 125+ eBioscience Super Bright antibody conjugates for the violet laser
  • Now available, the Super Bright Staining Buffer in 1000 test size (Cat. No. SB-4400-75)
• 12 new clones for flow cytometry including—
  • Anti-Human/Mouse Arginase 1 (clone A1exF5*)
  • Anti-Mouse CD163 (clone TNKUPJ)
  • Anti-Human AXL (clone DS7HAXL)
  • Anti-Human/Mouse Granzyme B (clone N4TL33)
  • Anti-Human ID2 (clone ILCID2)
  • Anti-Mouse AXL (clone MAXL8DS)

* For more information about the A1exF5 Arginase 1 clone, please see thermofisher.com/arginaseflowblog

See how the Super Bright antibody conjugates are being used by your colleagues, and check out these 3 references:

Intracellular staining of mouse bone marrow derived macrophages polarized to M1 or M2a, with Arginase 1 antibody. C57BL/6 mouse bone marrow derived macrophages were polarized for 24 hours with either IL-4 or LPS and IFN-gamma, and subsequently surface stained with F4/80 (clone BM8). Cells were then fixed and permeabilized with the IC Fixation and Permeabilization Buffer Set followed by intracellular staining with Arginase 1 (clone A1exF5). Total viable cells were used for analysis, as determined by Fixable Viability Dye staining. Consistent with the known expression pattern, Arginase 1 clone A1exF5 stains most M2a macrophages (right panel) and does not stain M1 macrophages (left panel).

Find antibodies for flow cytometry now

Oxygen deficiency of the cellular environment is referred to as hypoxia, and plays a role in many diseases. The Hypoxia Green Reagent for Flow Cytometry is an antibody-independent reagent used to detect low levels of oxygen levels in live cells and is a:

• Sensitive end-point assay
• Reagent that detects decreases in oxygen in live cells
• Fluorogenic, non-antibody based probe

Simultaneous detection of hypoxia and a cell surface marker in K562 leukemia cells. K562 cells, a human leukemia cell line that expresses CD71 (transferrin receptor), were incubated under normoxic (20% O₂) or hypoxic (1% O₂) conditions for 18 hours. Hypoxia Green Reagent was subsequently added to the cells and cells were incubated for an additional 3 hours. Cells were harvested, stained with PE-Cy7 anti-CD71 antibody, and analyzed using an Attune NxT Flow Cytometer configured with a 488 nm laser and a 530/30 nm emission filter to detect Hypoxia Green and a 561 nm laser and 780/60 nm emission filter to detect CD71.

Learn more about hypoxia green reagent for flow cytometry

The state of mitochondrial health can be assessed by detecting changes in the mitochondrial membrane potential. In a healthy cell with active mitochondria, tetramethylrhodamine, methyl ester (TMRM) is readily sequestered, thereby emitting a red-orange fluorescent signal. During apoptosis or upon treatment with a reagent such as carbonyl cyanide m-chlorophenyl hydrazone (CCCP), the mitochondrial membrane is depolarized and the TMRM signal diminishes.

Detection of mitochondrial potential changes in apoptotic cells. Jurkat cells, a human T-lymphocyte cell line, were treated with DMSO (control) or 500 nM staurosporine for 2 hours. Cells were subsequently stained with MitoProbe TMRM for 30 min at 37˚C, followed by a wash and subsequently l stained with Annexin V Pacific Blue conjugate. Data was acquired using an Attune NxT flow cytometer configured with a 561 nm laser and 585/16 nm emission filter for MitoProbe TMRM and 405 laser and 440/50 nm emission filter for Annexin V Pacific Blue conjugate. Staurosporine induced apoptosis, resulting in a mixed population of cells containing a population of healthy MitoProbe TMRM-positive cells as well as a population of apoptotic, Annexin V Pacific Blue-positive, MitoProbe TMRM-low cells.

Learn more about MitoProbe TMRM Assay Kit for Flow Cytometry

Viability determination using CyQUANT XTT Cell Viability Assay, Cell Proliferation Kit II (XTT), and MTT reagent. A549 cells were plated at various concentrations (see X-axis) and incubated. After an overnight incubation, manufacturer instructions were followed for the CyQUANT XTT Cell Viability Assay, Cell Proliferation Kit II (XTT) (by Sigma), and the MTT reagent. The XTT reagent containing the Electron Coupling Reagent was added and incubated for 4 hours at 37ºC in a CO₂ incubator. The absorbance was read at 450 nm and 660 nm using the Varioskan LUX. The specific absorbance signals were determined and the Signal to Background (wells with no cells) ratios were generated.

Learn more about CyQUANT XTT Cell Viability Assay

Two new reagents were added to our successful portfolio of Alexa Fluor Plus phalloidin conjugates to broaden the spectra characteristics of our product offering.

• Alexa Fluor Plus 405 Phalloidin – 405/450 Ex/Em
• Alexa Fluor Plus 750 Phalloidin – 758/784 Ex/Em

Fluorescent Alexa Fluor and Alexa Fluor Plus dye conjugates of phalloidin are widely used F-actin stains, with color choices across the full spectral range. These phalloidin conjugates provide researchers with fluorescent probes that are designed to be superior in brightness and photostability to all other spectrally similar conjugates tested.

Find more information and comparison with other phalloidin reagents

Learn more and find information and comparison with other HCS CellMask stains

The Invitrogen CytoVista reagents are optimized for 3D tissues, spheroids, organoids, and cell cultures of various thickness. Tissues sections 1 mm thick can be cleared for imaging in just 2 hours, while whole mouse brains of 8 mm thickness can now be cleared in 24 hours. Two comprehensive kits and a la carte reagents are available to suit different experimental needs:

• CytoVista 3D Cell Culture Clearing / Staining Kit is used for organoids/spheroids
• CytoVista Tissue Clearing / Staining Kit is used for samples up to 10 mm thick
   

Highlights

• Rapid clearing of fluorescent-labeled tissue for 3D imaging with minimal effect on detection sensitivity or sample morphology
• Workflow can be easily adapted to IHC workflow without additional equipment or instrument
• Cleared tissue can be reversed and processed for histology studies, such as hematoxylin and eosin (H&E) staining
• Compatible with most fluorophores, including dyes and fluorescent proteins
• Compatible with standard fluorescent imaging instruments such as wide-field microscope, high-content analyzers, confocal microscopes, and light sheet microscopes
• May be used to store and stabilize 3D tissue/cultures for weeks with minimal effect on sample morphology or fluorophore brightness and specificity

Learn more about CytoVista reagents

This series of courses was designed to provide engaging, interactive learning experiences relevant to protein and cell analysis application areas and topics. These free, self-paced, animated courses include knowledge checks and practical application exercises that enable you to demonstrate what you have learned by completing educational modules.

Series of courses:

• T Cell Stimulation and Proliferation
• Protein Sample Preparation
• Antibody Validation* (newly released)
• Cancer Spheroid and Organoid Models (coming soon)

All content is available 24 hours a day, 7 days a week, and is viewable from the convenience of your desk, tablet, or mobile device. See what it’s all about at thermofisher.com/elearningcourses

*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.

For research use purposes only

Webinar available on-demand, presented by Erik Portelius, Associate Professor, Neurochemistry, The Sahlgrenska Academy at the University of Gothenburg.

Immunoprecipitation in combination with mass spectrometry is a powerful tool for studying disease-associated proteins. Learn how Thermo Scientific's KingFisher instruments combined with our Dynabeads magnetic bead-based extraction, supports valuable research for Alzheimer's disease.

Watch the webinar on-demand

Designing a panel for flow cytometry is a highly complex process requiring a lot of time and effort. The Invitrogen Flow Cytometry Panel Builder makes panel design less intimidating and simpler to help you design your panel more efficiently, regardless of your panel design experience.

Other articles cover:

• Cell analysis using antibodies
• Tools for flow cytometry
• High-content imaging and analysis
• Protein and nucleic acid analysis
• Journal club—A view of the steady-state distributions of proteins within a cell

Read this issue at thermofisher.com/bp77

The subtopics section presents educational content by application, technique or research area, making it easier for you to find the educational resources you need. Each new subtopic page contains featured content as well as a search table that contains all related educational assets. Finding each asset is easy, just use the search box to type in a keyword and the table should display all relevant assets.

Cell analysis subtopics

• Cell imaging information
  • Fluorescence imaging information
  • Transmitted light/colorimetric imaging information
  • High-content analysis (quantitative imaging) information
  • Simple automated imaging information
• Cell structure information
• Cell viability and function information
  • Apoptosis information
  • Autophagy information
  • Cell viability, proliferation and cell cycle information
  • Endocytosis and phagocytosis information
  • Oxidative stress and hypoxia information
  • Ion indicators and ion channels information
• Cell counting information
• Cell analysis antibodies information
  • Immunofluorescence information
  • Immunohistochemistry information
• Labeling and detection information
• Flow cytometry information
• Cell analysis–related research areas

Start learning today at thermofisher.com/celllearning

Are your imaging experiments coming back with high background, again? Do you notice cross reactivity?

Watch this quick video to find a solution to publication quality images with Invitrogen cross- adsorbed Alexa Fluor secondary antibodies.

Learn more about cross-adsorbed secondary antibodies