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Proteostat

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Proteostat is a fluorescent dye designed to detect and quantify protein aggregation in a variety of applications. It binds selectively to misfolded or aggregated proteins, emitting a fluorescent signal that can be measured to assess the extent of protein aggregation in a sample.

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Lab products found in correlation

Proteostat assay kit, by Enzo Life Sciences (4 mentions) 710 confocal microscope, by Zeiss (3 mentions) Nitrocellulose membrane, by Merck Group (2 mentions) Dot blot apparatus, by Bio-Rad (2 mentions) Lysis buffer, by Enzo Life Sciences (2 mentions) Phosstop tablet, by Roche (2 mentions) Lsm 880, by Zeiss (2 mentions) Chloroquine, by Merck Group (1 mentions) Cyto id, by Enzo Life Sciences (1 mentions) 7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions) Sp8 microscope, by Leica camera (1 mentions) Zen 2, by Zeiss (1 mentions) Fluorshield with dapi, by Merck Group (1 mentions) Elyra ps 1, by Zeiss (1 mentions) Fluorobrite dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions) Bis ans, by Merck Group (1 mentions) Sypro orange, by Thermo Fisher Scientific (1 mentions) Brefeldin a bfa, by Merck Group (1 mentions) Lc3a b antibody, by Cell Signaling Technology (1 mentions) Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)

25 protocols using proteostat

1

Insulin Reduction Assay for PDI Activity

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The activity of human Protein Disulfide Isomerase was tested using the insulin reduction assay (ProteoStat™, Enzo life Sciences) with minor modifications. The catalyzed reduction of insulin was measured in the presence of tris(2-carboxyethyl)phosphine (TCEP) instead of DTT in order to avoid possible reaction of DTT with compounds. The reaction mixture contained 200 µM TCEP and 330 µM insulin in phosphate/EDTA buffer (pH 7.4). PDI was used at a final concentration of 1 μM. Inhibitor compounds at the indicated concentrations were included in the reaction mixture up to 100 µM. The reaction was initiated by the addition of TCEP in 96-well plates (100 µl final volume) for 30 min at 25º C in triplicates. The uncatalyzed reduction of insulin was subtracted from the reaction containing PDI.
Landeta C., Blazyk J.L., Hatahet F., Meehan B.M., Eser M., Myrick A., Bronstain L., Minami S., Arnold H., Ke N., Rubin E.J., Furie B.C., Furie B., Beckwith J., Dutton R, & Boyd D. (2015). Compounds targeting disulfide bond forming enzyme DsbB of Gram-negative bacteria. Nature chemical biology, 11(4), 292-298.
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2

Measuring Autophagy and Protein Aggregation

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Autophagy is a process that cells use to digest internal components and engulfed particles to recover nutrients. It is also a process that occurs during some forms of cell death or as a way to prevent cell death [26 ]. Activation of autophagy was assessed with a fluorescent dye (Cyto-ID™ (Enzo) that is quenched unless the dye is incorporated into cellular membranes formed by fusion of autophagosomes and lysosomes. Chloroquine (Sigma) was used as a specific inhibitor of lysosomal acidification to assess the autophagy initiation pathways involved. Cells can be induced to enter apoptosis when they are under the stress of the unfolded protein response (UPR). We used a red fluorescent rotor dye ProteoStat™ (Enzo) to stain protein aggregesomes and measured their relative concentrations by flow cytometry. A positive control supplied in the kit, MG-132, was used as an inducer of aggregesome formation because it inhibits proteasome activity.
Wagner R.D., Johnson S.J., Danielsen Z.Y., Lim J.H., Mudalige T, & Linder S. (2017). Polyethylene glycol-functionalized poly (Lactic Acid-co-Glycolic Acid) and graphene oxide nanoparticles induce pro-inflammatory and apoptotic responses in Candida albicans-infected vaginal epithelial cells. PLoS ONE, 12(4), e0175250.
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3

Thermal Stability Assay for Protein Unfolding

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DSF was performed using a fluorescent dye (ProteoStat, Enzo Life Sciences) and a real time PCR machine (Applied Biosystems 7500 Fast Real-Time PCR System). Samples were scanned from 25°C to 99°C, and the fluorescence intensity was monitored as a function of temperature. The temperature where rHA unfolded (characterized by a transition in the fluorescence signal) was determined from the second derivative plot, and expressed as midpoint transition temperature (Tm). The Tm is the temp where 50% of the molecules are in the unfolded state. The data were analyzed using the Applied Biosystems Protein Thermal Shift Software (Version 1.1).
Holtz K.M., Robinson P.S., Matthews E.E., Hashimoto Y., McPherson C.E., Khramtsov N., Reifler M.J., Meghrous J., Rhodes D.G., Cox M.M, & Srivastava I.K. (2014). Modifications of cysteine residues in the transmembrane and cytoplasmic domains of a recombinant hemagglutinin protein prevent cross-linked multimer formation and potency loss. BMC Biotechnology, 14, 1.
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4

Visualizing ECCA Aggregates in Live Cells

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To determine if ECCAs are aggregates live seeded 3D7-DiCre_pFIO+_PfCen1-GFP cells were stained with 1:1000 Hoechst 3342 and 1:3000 Proteostat (Enzo Life Sciences) in cRPMI for 30 min at 37°C. Live cells were imaged at 37°C using the Leica SP8 microscope with the Lighting module, enabling automated adaptive deconvolution. 264x264 pixel images were acquired using a 63x/1.4 N.A. oil immersion objective, GaAsP detectors, a pinhole setting of 0.6 airy units, a pixel size of 35.1 nm and z-stack of 7.28 μm with slices at 130 nm intervals. PfCen1-GFP and Proteostat were excited with a 488 nm laser, with GFP emission being measured between 490 to 550 nm and Proteostat emission at 600 to 700 nm. GFP signal bleed-through into the Proteostat range was negligible.
Voß Y., Klaus S., Lichti N.P., Ganter M, & Guizetti J. (2023). Malaria parasite centrins can assemble by Ca2+-inducible condensation. PLOS Pathogens, 19(12), e1011899.
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5

Protein Aggregation Detection in MEFs

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MEFs were cultivated on glass coverslips (Laboratory Glassware Marienfeld). 24 h after seeding, cells were heat stressed at 42 °C for 1 h or treated with 0.5 µM MG-132 for 48 h or 100 nM BafA1 for 48 h. Cells were fixed for 15 min with 4 % paraformaldehyde in PBS pH 7.4 and permeabilized with 0.5% (v/v) Triton X-100, 3 mM EDTA pH 8.0, and 5% goat serum in PBS for 30 min at room temperature and then stained with Proteostat® (Enzo Life Sciences, Inc.) at a dilution of 1:2000 in 1× assay buffer (ENZO) for 20 min. Cells were then mounted in Fluorshield with DAPI (Sigma). Fluorescence microscopy was performed using a Zeiss ELYRA PS.1 equipped with an LSM880 (Carl Zeiss, Oberkochen) with a 63 × oil immersion objective. Super-resolution images were generated by the Structured Illumination (SIM) technology. SIM confocal images were processed using the ZEN2.1 software (Carl Zeiss, Jena).
Furthmann N., Bader V., Angersbach L., Blusch A., Goel S., Sánchez-Vicente A., Krause L.J., Chaban S.A., Grover P., Trinkaus V.A., van Well E.M., Jaugstetter M., Tschulik K., Damgaard R.B., Saft C., Ellrichmann G., Gold R., Koch A., Englert B., Westenberger A., Klein C., Jungbluth L., Sachse C., Behrends C., Glatzel M., Hartl F.U., Nakamura K., Christine C.W., Huang E.J., Tatzelt J, & Winklhofer K.F. (2023). NEMO reshapes the α-Synuclein aggregate interface and acts as an autophagy adapter by co-condensation with p62. Nature Communications, 14, 8368.
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6

Quantifying Protein Aggregation in Hearts

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Protein aggregates were measured using Proteostat (Enzo, ENZ-51023) following the manufacturer’s instructions. For this assay left ventricle myocardial lysate (Cell Signaling lysis buffer) was obtained, protein concentration was assayed (BCA assay [Pierce]). Ten μg of protein was loaded into a 96-well microplate and protein aggregates were analyzed using the Proteostat assay kit (Enzo Life Sciences) following the manufacturer’s instructions. Background readings were subtracted from sample recordings and were normalized to wild-type values.
Qu J.H., Chakir K., Tarasov K.V., Riordon D.R., Perino M.G., Silvester A.J, & Lakatta E.G. (2024). Reprogramming of cardiac phosphoproteome, proteome, and transcriptome confers resilience to chronic adenylyl cyclase-driven stress. eLife, 12, RP88732.
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7

Single-Aggregate TIRF Imaging of Amyloid Proteins

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ProteoStat (ENZ-51023; Enzo Lifesciences) or AT630 (Ebba Biotech) was diluted 1:50 in PBS for stock aliquots and stored at -20 °C. This stock was further diluted 1:50 (ProteoStat) or 1:1,000 (AT630) for final single-aggregate TIRF imaging. Single-aggregate TIRF imaging was performed as described before (29 (link)). Briefly, aggregates were diluted to 700 nM (αS), 400 nM (tau), or 300 nM (Aβ) imaging concentrations with indicated fluorophores and incubated on a coverslip for 15 min. Samples were imaged on a home-built TIRF microscope. For super-resolution microscopy, samples were incubated in freshly prepared imaging buffer [PBS with 1 mg/mL glucose oxidase, 0.02 mg/mL catalase, 10% (wt/wt) glucose, and 100 mM methylamine] immediately prior to imaging.
Generation of the HEK293A cells expressing eGFP-tagged PSMD14 subunit is described in detail by Zhang et al. (64 (link)). Live-cell imaging of these cells incubated with 1 μM aggregates was conducted in FluoroBrite Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum.
Morten M.J., Sirvio L., Rupawala H., Mee Hayes E., Franco A., Radulescu C., Ying L., Barnes S.J., Muga A, & Ye Y. (2022). Quantitative super-resolution imaging of pathological aggregates reveals distinct toxicity profiles in different synucleinopathies. Proceedings of the National Academy of Sciences of the United States of America, 119(41), e2205591119.
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8

Visualizing Protein Aggregates in Neurons and Fibroblasts

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Cultured mice cortical neurons or human primary fibroblasts at 60% confluency were incubated with PROTEOSTAT® (Enzo LifeSciences) to detect protein aggregates.
Even A., Morelli G., Turchetto S., Shilian M., Bail R.L., Laguesse S., Krusy N., Brisker A., Brandis A., Inbar S., Chariot A., Saudou F., Dietrich P., Dragatsis I., Brone B., Broix L., Rigo J.M., Weil M, & Nguyen L. (2021). ATP-citrate lyase promotes axonal transport across species. Nature Communications, 12, 5878.
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9

Multiparametric Protein Aggregation Monitoring

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50 µmol L‐1 Bis‐ANS, 5X SYPRO Orange, 3 µmol L‐1 ProteoStat and 50 µmol L‐1 ThT were the final concentration in each well. ThT (Sigma Aldrich, Bangalore, India) and Bis‐ANS (Sigma Aldrich, Steinhelm, Germany) were solubilised into MilliQ water. SYPRO Orange (Life Technologies, Eugene, Oregon) was diluted using MilliQ water and ProteoStat (Enzo Life Sciences, Farmingdale, New York) was prepared based on the manual protocol. Concentration of mAb A was kept to 1 mg mL‐1 when using stressed mAb (mAb in cell culture supernatants had varying titres) with a final volume of 200 µL in each well (n = 3). The excitation and emission wavelengths (ex/em) for each dye were as follows: Bis‐ANS 390/450–600 nm, SYPRO Orange 495/550–700 nm, ProteoStat 530/560–700 nm and Thioflavin 430/460–600 nm. The assay was measured using a 96 well clear flat‐bottom micro plate (Corning, Kennebunk, Maine) with TECAN Infinite M200 series. The following parameters were used for fluorescence measurement: bottom reading, integration time 20 µs and 25 flashes. The data were analysed using OriginPro and smoothed using Savitzky–Golay smoothing filter with a polynomial of 2 and a smoothing factor of 15.
Oshinbolu S., Shah R., Finka G., Molloy M., Uden M, & Bracewell D.G. (2018). Evaluation of fluorescent dyes to measure protein aggregation within mammalian cell culture supernatants. Journal of Chemical Technology and Biotechnology, 93(3), 909-917.
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10

Antibody-based detection and analysis

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F12 (Coon’s modification) medium and BFA (brefeldin A) were purchased from Sigma–Aldrich. BFA was stored at −20°C as a 10 mg/ml stock in DMSO. Lactacystin was stored as a 5 mM stock at −20°C. MG-132 (Cell Signaling Technology) was stored at −20°C as a 10 mM stock in DMSO. FBS and newborn calf serum were purchased from Gemini Bio-Products. TaqSelect DNA polymerase was purchased from Lucigen. High-speed plasmid mini kits were from MidSci. HRP (horseradish peroxidase)-conjugated secondary antibodies and Western Lightning ECL reagent were purchased from Sigma–Aldrich and PerkinElmer respectively. Alexa Fluor®-conjugated secondary antibodies were purchased from Invitrogen. Monoclonal antibodies against the V5 epitope tag were from AbD Serotec or against mannosidase II were purchased from Covance. ProteoStat® was purchased from Enzo Life Sciences and cells were stained according to the manufacturer’s instructions. LC3A/B antibodies were from Cell Signaling Technology. Anti-MAL2 polyclonal antibodies were generated and affinity-purified as described previously [2 (link)]. Polyclonal antibodies against haptoglobin, HA321, CE9, rat serum albumin, APN (aminopeptidase N) and lysosomal glycoprotein 120 were provided by Dr A. Hubbard (Johns Hopkins University School of Medicine, Baltimore, MD, U.S.A.).
In J.G., Striz A.C., Bernad A, & Tuma P.L. (2014). Serine/threonine kinase 16 and MAL2 regulate constitutive secretion of soluble cargo in hepatic cells. The Biochemical journal, 463(2), 201-213.
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