Interferon-stimulated gene 15 (ISG15) encoded by the ISG15 gene is the first identified protein from a family of ubiquitin-like modifiers that share sequence and conformational homology to ubiquitin. The conjugation of ISG15 (Fig a, Fig b) to its target proteins is possible through a three-step process termed as ISGylation which is similar to ubiquitination. In analogy to the ubiquitin system, ISG15 conjugation or ISGylation is mediated by an enzymatic cascade consisting of an E1 activating enzyme UBE1L (Fig c); an E2 conjugating enzyme UBE2L6 and E3 ligases HERC5 (Human) (Fig d)/ HERC6 (Mouse)/ EFP/ HHARI.
Unlike ubiquitination however, ISGylation does not appear to mark proteins for proteasomal degradation; its biological function remains largely undiscovered on account of only a few ISG15 target proteins being identified till now. Considering that type I interferons (IFNs) play critical roles in innate immunity responses and cancer development, ISG15 conjugation is likely to be relatedto IFN mediated immune responses.
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Fig a.
Fig b.
Fig c.
Fig d.
Detection of the free form of Human ISG15 and its conjugates in A549 cells treated with IFN-alpha using ABfinity Anti-ISG15 Human Monoclonal Antibody (Cat. No. 703131, 1:5000) in western blot (Fig a) and (Cat. No. 703654, 1:100) in Immunofluorescence (Fig b).
Detection of UBE1L in A549 and HeLa cells treated with IFN-beta using ABfinity Anti-UBE1L Oligoclonal Antibody (Cat. No. 712170, 1:250) in western blot (Fig c).
Detection of HERC5 in HeLa cells upon siRNA transfection using ABfinity Anti-HERC5 Monoclonal Antibody (Cat. No. 703675, 1:500) in western blot (Fig d).
Click in the table to find out more about or order the ABfinity antibodies for ISGylation
Introducing ChIP-grade ABfinity antibodies for mutant histone variants
Histone variants differ from their canonical counterparts in one or a few amino acids that confer specific structural and functional features to these variants. One such histone variant, histone H3.3 is deposited in a replication-independent manner and is associated with transcriptionally active chromatin. Recurrent driver mutations in the histones, predominantly H3.3, trigger genome-wide epigenetic changes, and are common features of certain cancers. These mutations include K27M, G34R/G34V, and K36M. The substitution of glycine at position 34 (G34) modulates methylation at K36, and thus affects transcriptional elongation.
Data from our ChIP-grade monoclonal and oligoclonal recombinant ABfinity H3.3G34R antibodies (Cat. No. 703360, 711969) antibodies are shown below.
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Antibody specificity in WB and ChIP, using H3.3G34R overexpressing HEK-293T cells and in SNAP-ChIP (Sample Normalization and Antibody Profiling). Testing of ABfinity Anti-H3.3G34R antibodies (Cat. No. 703360 & 711969) in western blot (A), SNAP-ChIP (B) and ChIP assay (C). Both antibodies react to only H3.3G34R proteins in WB and SNAP-ChIP, and show enrichment only at transcriptionally active genes. Asterisk (*) indicates a non-specific band observed at ~55 kDa.
Find these and other antibodies for your research using our antibodies search tool at thermofisher.com/antibodies.
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 out our search experience that:
Helps you quickly identify the most referenced clone
Shows you images directly from the literature
Allows you to click directly through to the articles which have used the antibody that is of interest to you
Need help finding and selecting the right antibodies for your research? Design your flow cytometry panel now with the Invitrogen Flow Cytometry Panel Builder.
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F4/80 Monoclonal Antibody in flow cytometry. C57BL/6 mouse resident peritoneal exudate cells were surface stained with F4/80 Monoclonal Antibody, eFluor 450 (Cat. No. 48-4801-82). The cells were then fixed and permeabilized using the Intracellular Fixation and Permeabilization Buffer Set (Cat. No. 88-8824-00) and intracellularly stained with either 0.25 µg of Rat IgG1 kappa Isotype Control, APC (Cat. No. 17-4301-82) (left) or 0.25 µg of RELM alpha Monoclonal Antibody, APC (right). All cells were used for analysis.
Invitrogen ProQuantum High-Sensitivity Immunoassay Kits - 35 now available
Invitrogen ProQuantum high-sensitivity immunoassays are a platform innovation that provides researchers with an easy-to-run, high-performance assay, with no proprietary instrument to purchase. By utilizing proximity-based amplification technology, we have combined the analyte specificity of high affinity antibody–antigen binding with the signal detection and amplification capabilities of qPCR, to achieve a highly sensitive protein quantitation assay. With the ability to detect even lower levels of protein than traditional methods and with very low sample consumption, you can get the most out of your precious or limited samples.
High sensitivity—detect low levels of protein with greater sensitivity than traditional methods
Broad dynamic range—≥5 orders of magnitude, minimizing sample dilutions to ensure they fall within the range
Low sample consumption—use 2–5 μL of sample (for example, 2 μLvs. 150 μL for triplicate wells with other methods)
Fast, easy workflow—2 hours from sample to answer, no wash steps, and a single 1-hour incubation
Intuitive cloud-based software—comprehensive data analysis and statistical groupwise comparison
How ProQuantum Immunoassays work
ProQuantum immunoassays utilize a matched pair of target-specific antibodies, each conjugated to a DNA oligonucleotide. During antibody–analyte binding, the two DNA oligos are brought into close proximity, which then allows ligation of the two strands and subsequent creation of a template strand for amplification.
This immunoassay leverages the sensitivity and large dynamic range of Applied Biosystems TaqMan. qPCR technology. The qPCR data file can be imported into the ProQuantum software (apps.thermofisher.com/apps/proquantum) for easy standard curve analysis and results.
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Two-step process. (A) Antibody conjugates binding to target (1-hour incubation). (B) Ligation and amplification of signal (in qPCR instrument).
See the complete list of available protein targets and those coming soon and learn more at thermofisher.com/proquantum.
New AlamarBlue HS and PrestoBlue HS Cell Viability Reagents
The alamarBlue and PrestoBlue HS (high sensitivity) reagents are a new generation of assays that offer superior performance than the first generation of alamarBlue and PrestoBlue reagents. These assays detect cell viability based on cellular redox potential (conversion of resazurin to resofurin) and are compatible with fluorescence- or absorbance-based instruments.
Key advantages:
Removal of contaminations from resazurin — displays a >50% reduction in background fluorescence
Signal-to-background ratio increased by >100% — resulting in large dynamic range
Highly sensitive with a linear response — detects as few as 10 cells per well
Convenient add-and-read format — no mixing, no washing, no cell lysis, and compatible with either fluorescence- or absorbance-based instrumentation
Comparison between alamarBlue HS and PrestoBlue HS
alamarBlue HS
PrestoBlue HS
Contains highly purified resazurin, resulting in low background and higher signal-to-background ratio
Yes
Yes
Works with diverse cells types (mammalian, fungal, plant, bacterial, etc.)
Yes
Optimized for mammalian cells
Yes
Longer experiments or higher cell densities
Yes
Faster determination of viability (~10 minutes)
Yes
Performance comparison of alamarBlue cell viability reagents. A549 (left panel) or HeLa (right panel) cells were seeded at 5,000 cell/well and incubated overnight to allow for attachment. Only complete media was added to the Background wells. A 10 µL aliquot of alamarBlue HS (Thermo Fisher Scientific), alamarBlue (Thermo Fisher Scientific) or alamarBlue (Bio-Rad) reagent was added to the wells containing 100 µL of complete growth media and the attached cells. At various time points post-addition of the alamarBlue cell health reagents the fluorescence was measured using at excitation 560nm and an emission of 590nm. The Signal to Background ratio was generated by subtracting the Background fluorescence (media and alamarBlue cell health reagent) from the Signal fluorescence (cells, media and alamarBlue cell health reagent). Within 30 minutes the increase in the Signal to Background ratio for the alamarBlue HS is clearly visible and increases over time for both cell lines. After four hours of incubation the A549 cells treated with alamarBlue HS displays a Signal to Background ratio of >200-fold; when compared to the ratio for alamarBlue (from Thermo Fisher or Bio-Rad) of 20-fold this is a >100% increase in the signal to background ratio for the alamarBlue HS versus the alamarBlue reagent.
If you are currently using alamarBlue and PrestoBlue reagents (DAL1025, DAL1100, A13261, A13262), you can try these new HS versions.
CellEvent Senescence Kits for imaging and flow cytometry
The CellEvent Senescence Reagent kits are optimized for the fluorescent detection of senescent cells. Upregulation and accumulation of the β-galactosidase enzyme (β-gal) is commonly used as a biomarker for senescent cells. This hydrolase enzyme resides in lysosomes and converts β-galactosides into monosaccharides under acidic pH conditions.
The kits' fluorescent probe contains two galactoside moieties, making it specific for β-galactosidase. The enzymatically cleaved product is retained within the cell due to covalent binding of intracellular proteins and emits a fluorogenic signal that has absorption/emission maxima of 490/514 nm.
These kits offer:
Reliable and quick fluorescence detection of senescent cells
Simply add, incubate, and detect using standard Alexa Fluor 488/FITC filter sets
Multiplexing – fluorescent senescence probe can be multiplexed
T47D cells were treated with 5 µM palbociclib and fixed with 4% paraformaldehyde on days 0, 2, 6, 8 and 14 post-treatment. After fixation the cells were stain with either CellEvent Green Senescence Probe for 90 minutes or overnight with X-Gal. The X-Gal was images (top row) were captured in using brightfield and the fluorescence signal (bottom row) was captured using the standard FITC/Alexa Fluor 488 settings.
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T47D cells were treated with 5 µM palbociclib (senescent) for 16 days or untreated (cycling). After treatment the cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature; then sequentially stained with CellEvent Green Senescence Probe for 90 minutes in 37ºC incubator with no CO2 and then stained overnight with X-Gal. The X-Gal was imaged using brightfield (Panels A and C) and fluorescence (Panels B and D) using the standard FITC/Alexa Fluor 488 settings.
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Cyclin A2 and B1 expression decreases with the onset of senescence. Senescence was induced in T47D, human epithelial cells. Cycling T47D cells progress through the various cell cycle stages as indicated by the expression of cyclin A2 and B1 (A). A lack of expression of cyclins (B) demonstrates cell cycle arrest in senescent cells.
New cloud library protocols for antibody workflows in microplate readers
Antibody-related applications are now even more straightforward with the aid of the Cloud Library in the Thermo Scientific SkanIt Software 6.0, which controls all Thermo Scientific microplate readers. The SkanIt Cloud Library is a collection of validated sessions, with real assay data and calculations, that all users can access for performing immediate measurements, or as templates to create their own assay protocols. With SkanIt Software 6.0, the Cloud Library is now available in a redesigned format, making it simpler to search for protocols by instrument, detection technology, plate format or other relevant search terms. The library is also expanded to cover more than 130 sessions. Each Cloud Library session has complete measurements with the required calculation and graph processing steps as well as representative data from real assays. Instructions for running the assays are also provided.
Ready-made cloud sessions are available for quick estimations of concentrations of antibodies using absorbance measurements at 280 nm. Assays can be selected to be performed with the Thermo Scientific μDrop Plate (for micro volumes) or with 96-well plates, using Thermo Scientific Multiskan Sky Spectrophotometer or Varioskan LUX Multimode Microplate Reader. When characterizing dye-conjugated antibodies, fast fluorometric titrations can be carried out with the dedicated protocols of Thermo Scientific Varioskan LUX, Fluoroskan or Fluoroskan FL microplate readers. Spectral measurements are also included with Varioskan LUX, for antibody validation purposes.
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Fluorometric titration (standard curve) for Alexa Fluor 647 conjugates.
Alexa Fluor Plus secondary antibodies- new testing data
Visit the updated web page for these secondary antibody conjugates launched at the end of 2018. These innovative secondary antibody conjugates combine the performance of Alexa Fluor Plus dyes with the cross-adsorbed secondary antibodies to deliver greater sensitivity and best signal-to background ratio. Review the testing data or search for the antibodies you need for your application and/or multiplexing.
Comparison product data is now available, along with an easy to use Alexa Fluor Plus secondary antibody ordering table.
Invitrogen Alexa Fluor Plus Secondary Antibody products Click on the catalog number below to choose.
Do you have questions about macrophages? What are they? What do they do? Read the blog post where we talk with our expert, Dariusz Stepniak, Ph.D., a Staff Scientist in R&D at Thermo Fisher Scientific to answer these questions. Additional topics covered include a discussion about macrophage classification, the difference between M1 and M2 macrophages and key markers to consider when characterizing macrophages. A few key references about macrophages are also provided.
Read the blog: New insights through the RELM alpha antibody
The Invitrogen anti-mouse RELM alpha monoclonal antibody (clone DS8RELM) recognizes RELM alpha, also known as resistin-like alpha or FIZZ1 a small cytokine produced mostly by macrophages. This antibody is particularly valuable to the scientists who aim to better understand the process of macrophage polarization. For more information about the DS8RELM clone, please read the interview with Dariusz Stepniak, Ph.D., a Staff Scientist in R&D at Thermo Fisher Scientific, who was responsible for the development of this new antibody.
