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Anti alix

Manufactured by Santa Cruz Biotechnology
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Anti-Alix is a laboratory reagent used for the detection and quantification of the Alix protein, a key component of the endosomal sorting complexes required for transport (ESCRT) machinery. It is designed to facilitate the analysis of Alix expression and localization in various biological samples.

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Lab products found in correlation

Anti cd63, by Santa Cruz Biotechnology (15 mentions) Anti tsg101, by Santa Cruz Biotechnology (13 mentions) Anti gapdh, by Santa Cruz Biotechnology (7 mentions) Anti cd81, by Santa Cruz Biotechnology (7 mentions) Anti cd63, by BD (6 mentions) Anti tsg101, by Abcam (5 mentions) Anti cd9, by Santa Cruz Biotechnology (5 mentions) Anti cd63, by Abcam (5 mentions) Anti β actin, by Merck Group (5 mentions) Pvdf membrane, by Merck Group (5 mentions) Anti tsg101, by Novus Biologicals (4 mentions) Anti cd9, by BD (4 mentions) Anti calnexin, by Santa Cruz Biotechnology (4 mentions) Anti hsp90, by Santa Cruz Biotechnology (3 mentions) Anti hsp70, by Santa Cruz Biotechnology (3 mentions) Anti actin, by Santa Cruz Biotechnology (3 mentions) Nitrocellulose membrane, by Bio-Rad (3 mentions) Anti calnexin, by Abcam (3 mentions) Mini protean tgx precast protein gel, by Bio-Rad (3 mentions) Clarity western ecl substrate, by Bio-Rad (3 mentions)

56 protocols using anti alix

1

Quantifying Extracellular Vesicle Proteins

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The concentration of total protein in EV samples and cells lysed in RIPA buffer was determined using the NanoOrange™ protein quantitation kit (#N6666, ThermoFisher Scientific, Eugene, OR, USA) according to the manufacturer’s recommendations using a SpectraMax M5e microplate reader (Molecular Devices, LLC., San Jose, CA, USA). Immunoblotting was performed according to the previously described procedure [25 (link)] with the differences that 5 µg of total protein was applied to SDS-PAGE and proteins were visualized with SuperSignal™ West Femto Maximum Sensitivity Substrate (#34095, ThermoFisher Scientific, Rockford, IL, USA). The following primary and secondary antibodies and dilutions were used: anti-Alix (#sc-271975, 1:500; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Flotillin-2 (#3436S, 1:1000; Cell Signaling Technology, Topsfield, MA, USA), anti-CD9 (#13174, 1:2000; Cell Signaling Technology), anti-TSG-101 (ab125011, 1:5000; Abcam, Cambridge, UK), anti-PCNA (#sc-7907, 1:500; Santa Cruz Biotechnology), anti-Stomatin (#sc-134554, 1:500; Santa Cruz Biotechnology), anti-mouse goat polyclonal antibodies (#2367, 1:5000; Cell Signaling Technology); and anti-rabbit goat polyclonal antibodies (#29902, 1:80,000; Cell Signaling Technology).
Skryabin G.O., Vinokurova S.V., Galetsky S.A., Elkin D.S., Senkovenko A.M., Denisova D.A., Komelkov A.V., Stilidi I.S., Peregorodiev I.N., Malikhova O.A., Imaraliev O.T., Enikeev A.D, & Tchevkina E.M. (2022). Isolation and Characterization of Extracellular Vesicles from Gastric Juice. Cancers, 14(14), 3314.
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2

Western Blot Analysis of EV Markers

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Ten micrograms of EVs or cell lysates were resuspended in Laemmli sample buffer and separated by electrophoresis on 10% SDS-polyacrylamide gels. Gels were electroblotted onto nitrocellulose paper (Hybond-ECL nitrocellulose membrane, 0.2-μm transfer membrane; GE Amersham Life Sciences, USA) and blocked overnight with 5% non-fat dry milk in Tris buffer saline (B-TBS). Blots were incubated overnight at 4°C with mouse anti-CD63, anti-TSG-101, anti-ALIX, anti-HSP-70, or anti-HSP-90 antibodies (Santa Cruz Biotechnology, TX, USA). Finally, blots were washed and incubated with an anti-mouse HRP-conjugated or an anti-rabbit HRP-conjugated serum. Membranes were revealed with the ECL kit (GE, Amersham Life Sciences, USA) following the manufacturer’s instructions. The GE Healthcare, Image Quant TM-RT ECL, Version 1.0 software was used to detect specific proteins (25 (link)).
Menay F., Herschlik L., De Toro J., Cocozza F., Tsacalian R., Gravisaco M.J., Di Sciullo M.P., Vendrell A., Waldner C.I, & Mongini C. (2017). Exosomes Isolated from Ascites of T-Cell Lymphoma-Bearing Mice Expressing Surface CD24 and HSP-90 Induce a Tumor-Specific Immune Response. Frontiers in Immunology, 8, 286.
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3

Western Blot Antibody Validation

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The nitrocellulose membranes were blocked and antibodies were applied in 5% milk in TRIS-buffered saline/Tween-20 (TBST, 0.1M TRIS-HCl, 0.9% [w/v] NaCl, 0.1% [v/v] Tween-20, pH 7.5). The antibodies used were anti-NF45/ILF2 (Bethyl, A303-147A-M), anti-DHX9 (Bethyl, A300-855A-M), anti-Matrin-3 (Bethyl, A300-591A-M), anti-hnRNPA1 (Novus, NB100-672), anti-Caprin-1 (Proteintech, 15112-1-AP), anti-GST (Santa Cruz, sc-459), anti-DDX3X (Sigma, HPA001648), anti-actin (Santa Cruz, sc-1616), anti-FLAG (Sigma, A8592), anti-Alix (Santa Cruz, sc-49268), anti-TSG101 (GeneTex, GTX70255), anti-CD63 (Santa Cruz, sc-15363), and anti-Histone H3 (Cell Signaling Technology, 9715). The immunoblotting images were acquired with the Chemidoc MP Imaging System (Bio-Rad) and quantified with the Image Lab software (Bio-Rad).
Kamelgarn M., Chen J., Kuang L., Arenas A., Zhai J., Zhu H, & Gal J. (2016). Proteomic analysis of FUS interacting proteins provides insights into FUS function and its role in ALS. Biochimica et biophysica acta, 1862(10), 2004-2014.
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4

Immunofluorescence Analysis of Exosome Markers

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Cells were plated in a Labtek Chamber slide at a density of 2 × 104 cells/mL After three days, when the cells had reached near confluency, medium was removed from the cells and they were washed with PBS. After fixation with methanol 100% for 5 min, hPDLSC were incubated over night at 4 °C with primary antibodies. The antibodies used in this study include the following: anti-CD9 (monoclonal mouse, IgG1k, 1:1000, BioLegend, San Diego, CA, USA), anti-CD63 (monoclonal mouse, IgG1k, 1:500, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and anti-ALIX (monoclonal mouse, IgG1k,1:500, Santa Cruz Biotechnology, Inc., Dallas, TX, USA). For negative controls, the primary antibody was replaced by non-immune serum (mouse IgG, 1:500, Sigma-Aldrich, Saint-Louis, MO, USA). Then a secondary goat anti mouse AlexaFluor488 antibody (LSBio, Seattle, WA, USA) was applied for 1 h at RT. Counterstaining was performed using Hoechst-dye (1:2000, Sigma-Aldrich, Saint-Louis, MO, USA). Observations were performed using an Upright Widefield Microscope Leica DMRXA2 (Leica Microsystemes, Wetzlar, Germany).
Novello S., Tricot-Doleux S., Novella A., Pellen-Mussi P, & Jeanne S. (2022). Influence of Periodontal Ligament Stem Cell-Derived Conditioned Medium on Osteoblasts. Pharmaceutics, 14(4), 729.
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5

Comprehensive Immune Signaling Antibody Protocols

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Puromycin was purchased from Sigma‐Aldrich. GAPDH, β‐actin, GFP, OFP, SHP2, p‐LCK, LCK, p‐AKT (Thr308), AKT, p‐ZAP70, and ZAP70 antibodies for western blot were purchased from Abmart. FGL1 antibodies were purchased from Santa Cruz Biotechnology Inc. Antibodies, including human PD‐L1 and PD‐1 were purchased from Invitrogen. Human LAG‐3 and Na+K+ATPase antibodies were purchased from Cell Signaling Technology and Santa Cruz Biotechnology Inc. respectively. Marker antibodies for exosomes, including anti‐CD9, anti‐CD63, and anti‐ALIX, were purchased from Santa Cruz Biotechnology Inc, and anti‐CD81 from System Biosciences. Wheat Germ Agglutinin (WGA) Alexa Fluor 488 and 350 dyes were purchased from Thermo Scientific. Ficoll Paque Plus used for isolating peripheral blood mononuclear cells (PBMC) cells was purchased from GE Healthcare. Staining antibodies, including CD3, CD4, CD8, CD25, and Foxp3 for FACS analysis were purchased from Biolegend Inc.
Tsai H., Wu Y., Liu X., Xu Z., Liu L., Wang C., Zhang H., Huang Y., Wang L., Zhang W., Su D., Khan F.U., Zhu X., Yang R., Pang Y., Eriksson J.E., Zhu H., Wang D., Jia B., Cheng F, & Chen H. (2021). Engineered Small Extracellular Vesicles as a FGL1/PD‐L1 Dual‐Targeting Delivery System for Alleviating Immune Rejection. Advanced Science, 9(3), 2102634.
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6

Cell Culture and Antibody Protocols

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Homo sapiens HepG2 (ATCC HB-8065) and Hela (ATCC CCL-2) cells were cultured as a monolayer at 37 °C in complete Dulbecco’s modified Eagle’s medium (Gibco, USA) supplemented with 10% (vol/vol) fetal bovine serum (FBS) (Gibco, USA) under a humidified atmosphere containing 5% CO2. Fathead minnow (FHM) cells (ATCC CCL-42) were grown in M199 medium supplemented with (Gibco, USA) 10% (vol/vol) FBS at 27 °C.
Rabbit and mouse polyclonal antisera against TFV MCP were previously generated in our laboratory. Rabbit monoclonal antibody against the MYC tag and anti-Nedd4 antibody was purchased from Abcam (UK). Rabbit monoclonal anti-GAPDH antibody was obtained from CST (BRD). Mouse monoclonal anti-Tsg101, anti-Alix, anti-VPS4B, and rabbit polyclonal anti-VPS4 antibody were acquired from Santa Cruz (USA). Anti-rabbit/mouse IgG (H+L)-HRP conjugates were purchased from Promega (USA). All antibodies were used according to the manufacturers’ instructions.
Mi S., Qin X.W., Lin Y.F., He J., Chen N.N., Liu C., Weng S.P., He J.G, & Guo C.J. (2016). Budding of Tiger Frog Virus (an Iridovirus) from HepG2 Cells via Three Ways Recruits the ESCRT Pathway. Scientific Reports, 6, 26581.
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7

Comprehensive Protein Extraction and Analysis

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Total cell extracts (TCEs) were prepared in denaturing buffer (50 mM Tris–HCl pH 8.0, 600 mM NaCl, 0.5% sodium deoxycholate, 1% NP40 0.1% sodium dodecyl sulfate, and 1 mM EDTA) supplemented with protease and phosphatase inhibitors (Roche). Proteins were resolved using precast Bolt Novex Bis–Tris Gels 4–12% (Life Technologies), transferred to nitrocellulose membranes (Bio-Rad), and immunoreactivity was determined using ECL (Amersham). Acquisition and densitometric analysis of images were obtained using Image Lab software (Bio-Rad). Employed Abs were as follows: anti-HIPK2 (rat monoclonal Ab C5C6 kindly provided by M. L. Schmitz); anti-Spastin (1:100; #sc-53443), anti-GAPDH (1:1,000; #sc-32233), anti–α-tubulin (1:1,000; #sc-5286), anti-actin (1:1,000; #sc-47778), anti-ALIX (1:200; #sc-53538), anti-p62 (1:1,000; #28359), anti-lamina A/C (1:1,000; #sc-20680) by Santa Cruz Technology; anti-CEP55 (1:1,000; #00055165-A01), and anti-NBR1 (1:500; #H00004077-B01P) by Abnova; anti-TSG101 (1:500: #ab30871) and anti-H2B (1:1,000; #ab52484) by Abcam; anti-LC3 (1:1,000; #L8918 by Sigma–Aldrich) recognizing both the cytosolic LC3-I and lipidated LC3-II forms of LC3. Anti–horseradish peroxidase–conjugated goat anti–mouse #7076, anti–rabbit #7074, and anti–rat #7077 (Cell Signaling Technology).
Sardina F., Monteonofrio L., Ferrara M., Magi F., Soddu S, & Rinaldo C. (2020). HIPK2 Is Required for Midbody Remnant Removal Through Autophagy-Mediated Degradation. Frontiers in Cell and Developmental Biology, 8, 572094.
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8

Immunoblot Analysis of Urinary Proteins

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Immunoblot analysis was performed as described previously [16 (link)]. Briefly, each urine sample was loaded with an equal amount of creatinine per lane, and separated by SDS-PAGE. For the detection of each protein, we used the following primary and secondary antibodies: anti-AQP1 (catalog no. sc-20810, Santa Cruz Biotechnology, Santa Cruz, Dallas, TX), anti-AQP2 (catalog no. sc-9882, Santa Cruz Biotechnology), anti-TSG101 (catalog no. ab83–100, Abcam, Cambridge, UK), anti-Alix (catalog no. sc-49268, Santa Cruz Biotechnology), anti-rabbit IgG (catalog no. 7074, Cell Signaling Technology, Danvers, MA), anti-mouse IgG (catalog no. 1858413, Thermo Fisher Scientific, Waltham, MA), and anti-goat IgG (catalog no. P0449, Dako Japan, Tokyo, Japan). Antibody-antigen interactions were visualized using the SuperSignal West-Femto Chemiluminescence detection system (Thermo Fisher Scientific, Waltham, MA). Quantitative analysis of the resulting bands was performed using the WinRoof software package version 5.7 (Mitani, Tokyo, Japan).
Oshikawa-Hori S., Yokota-Ikeda N., Sonoda H, & Ikeda M. (2019). Urinary extracellular vesicular release of aquaporins in patients with renal transplantation. BMC Nephrology, 20, 216.
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9

Western Blot Analysis of Exosomal Proteins

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Cells and exosomes were decomposed by using 2 × SDS-lysis buffer. The protein samples were loaded into the SDS-PAGE gel and shifted onto a 0.22-μm nitrocellulose membrane (Axygen, USA). Membranes were blocked with 5% skim milk and then embraced with the primary antibodies overnight at 4°C. The next day, the membranes were washed and incubated with a second antibody for 1 h at room temperature. Finally, the membranes were visualized with an Enhanced Chemiluminescence (ECL) Detection Kit (Millipore, Billerica, USA) and by using ImageQuant LAS 4000 Mini (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The primary antibodies used were listed as follows: anti-HIF-1α (1:1,000; Cell Signaling Technology, USA); anti-ACTB (1:5,000; Cell Signaling Technology, USA); anti-TSG101 (1:1,000; Proteintech, USA); anti-ALIX (1:500; Santa Cruz, USA); anti-CD63 (1:1,000; Abcam, UK); anti-AKT, anti-p-AKT, anti-ERK1/2, anti-p-ERK1/2, anti-p38MAPK, and anti-p-p38MAPK (1:1,000; Cell Signaling Technology, USA); and anti-AMOTL2 (1:500; Proteintech, USA).
Guo Z., Wang X., Yang Y., Chen W., Zhang K., Teng B., Huang C., Zhao Q, & Qiu Z. (2020). Hypoxic Tumor-Derived Exosomal Long Noncoding RNA UCA1 Promotes Angiogenesis via miR-96-5p/AMOTL2 in Pancreatic Cancer. Molecular Therapy. Nucleic Acids, 22, 179-195.
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10

Immunological Antibody Characterization Protocol

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The table below describes the commercial antibodies used, their catalog numbers, and their company.
Western blot and immunofluorescence antibodies.
Anti-Alix (sc-49267)(Santa Cruz Biotechnology)
Anti-β-actin (MA1-91399)(Thermo Fisher Scientific)
Anti-β-actin (sc-47778)(Santa Cruz Biotechnology)
Anti-CD4 (2009-09)(Novocastra)
Anti-CD63 (sc-15363)(Santa Cruz Biotechnology)
Anti-EEA1 (610456)(BD Biosciences)
Anti-Flotillin1 (ab41927)(Abcan)
Anti-GAPDH (G9545)(Sigma-Aldrich)
Anti-GFPGift from R. Hedge (MRC)
Anti-ΗΑ (H3663)(Sigma-Aldrich)
Anti-HLA-A (15240-1-AP)(Proteintech)
Anti-IFITM1 (60074-1-Ig)(Proteintech)
Anti-IFITM2 (66137-1-Ig)(Proteintech)
Anti-IFITM3/2 (11714-1-AP)(Proteintech)
Anti-Lat1 (sc-54229)(Santa Cruz Biotechnology)
Anti-TfR (136800)(Thermo Fisher Scientific)
Anti-Tsg101 (976126)(BD Bioscencies)
Anti-Nef (2949)(NIH AIDS Reagent Program)
Anti-Neurexin1 (ab77596)(BD Bioscencies)
Anti-Syntenin1 (ab133267)(Abcan)
Anti-IgG Mouse-HRP(GE)
Anti-IgG Rabbit-HRP(GE)
Anti-IgG Goat-HRP(GE)
Anti-IgG Mouse-Alexa 594(Thermo Fisher Scientific)
Anti-IgG Rabbit-Alexa 647(Thermo Fisher Scientific)
da Silva-Januário M.E., da Costa C.S., Tavares L.A., Oliveira A.K., Januário Y.C., de Carvalho A.N., Cassiano M.H., Rodrigues R.L., Miller M.E., Palameta S., Arns C.W., Arruda E., Paes Leme A.F, & daSilva L.L. (2023). HIV-1 Nef Changes the Proteome of T Cells Extracellular Vesicles Depleting IFITMs and Other Antiviral Factors. Molecular & Cellular Proteomics : MCP, 22(12), 100676.
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