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Veriblot

Manufactured by Abcam
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Sourced in United Kingdom, United States

VeriBlot is a secondary antibody designed for use in Western blotting applications. It is a high-quality, affinity-purified antibody that specifically binds to the primary antibody used in the assay, allowing for the detection of target proteins.

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Lab products found in correlation

Nupage 4 12 bis tris, by Thermo Fisher Scientific (2 mentions) Hrp conjugated secondary antibody, by Agilent Technologies (2 mentions) Amersham imager 600, by GE Healthcare (2 mentions) Tris acetate gel, by Thermo Fisher Scientific (2 mentions) Nupage sample reducing agent, by Thermo Fisher Scientific (2 mentions) Nupage loading buffer, by Thermo Fisher Scientific (2 mentions) Imagequant 800, by GE Healthcare (2 mentions) Super signal west fempto chemiluminescent ecl, by Thermo Fisher Scientific (2 mentions) Amersham imager, by Cytiva (2 mentions) Surebeads protein a magnetic beads, by Bio-Rad (2 mentions) Hrp conjugated goat anti mouse or rabbit igg, by Jackson ImmunoResearch (2 mentions) Abs16, by Merck Group (1 mentions) Light chain specific secondary antibody, by Jackson ImmunoResearch (1 mentions) Anti ha, by Merck Group (1 mentions) Anti phospho histone h2ax, by Cell Signaling Technology (1 mentions) Anti pak4, by Cell Signaling Technology (1 mentions) Anti rela, by Santa Cruz Biotechnology (1 mentions) Secondary anti rabbit igg, by Cell Signaling Technology (1 mentions) Immobilon p polyvinylidene difluoride pvdf membrane, by Merck Group (1 mentions) Novex nupage sds page gels, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

VeriBlot for IP Detection Reagent (13 citations) VeriBlot for IP Detection Reagent (HRP) (7 citations) VeriBlot secondary antibody (5 citations) VeriBlot IP Detection Reagent (4 citations) VeriBlot for IP secondary antibody (4 citations) Veriblot-HRP (4 citations) Veriblot reagent (4 citations) Anti-mouse IgG VeriBlot for IP secondary antibody (3 citations) Veriblot anti-rabbit HRP (2 citations) VeriBlot for IP Dectection Reagent (2 citations)

15 protocols using veriblot

1

Cardiac Protein Localization and Quantification

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Animals were sacrificed by cervical dislocation, and hearts were flushed with ice-cold PBS and snap-frozen in liquid nitrogen. Immunofluorescence from frozen tissue was performed as described (45 (link)) using FLNC antibody RR90 (10 (link)), pan-cadherin antibody (Sigma), and myomesin antibody B4 (Developmental Studies Hybridoma Bank). Immunoblotting from total protein extracts was performed as described (45 (link)) using anti-HspB1 (2442, Cell Signaling Technology), anti-PHspB1 (Ser82) (2401, Cell Signaling Technology), anti-GAPDH (glyceraldehyde phosphate dehydrogenase) (ABS16, Merck), and FLNC [R1899; MRC PPU (Medical Research Council Protein Phosphorylation and Ubiquitinylation Unit) Reagents and Services, University of Dundee] antibodies. IP was performed as described (45 (link)) using 5 μg of FLNC (R1899) antibody. Blotting for HspB1 (2442, Cell Signaling Technology) was performed using “VeriBlot” (ab131366, Abcam) as secondary horseradish peroxidase conjugate.
Collier M.P., Alderson T.R., de Villiers C.P., Nicholls D., Gastall H.Y., Allison T.M., Degiacomi M.T., Jiang H., Mlynek G., Fürst D.O., van der Ven P.F., Djinovic-Carugo K., Baldwin A.J., Watkins H., Gehmlich K, & Benesch J.L. (2019). HspB1 phosphorylation regulates its intramolecular dynamics and mechanosensitive molecular chaperone interaction with filamin C. Science Advances, 5(5), eaav8421.
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2

Immunoprecipitation of Cx45 in MDCK Cells

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MDCK cells stable expressing either Cx45 WT or 6E were seeded in either 10 or 15 cm plates and grown to confluence. Cells were rinsed 2x with 1x TBS + 1mM EDTA and then scraped up an either 0.5 or 1.0 mL of IP lysis buffer (25mM Tris, 150mM NaCl, 0.25% sodium deoxycholate, 0.05% SDS, 1% TX-100 plus protease and phosphatase inhibitors). Cells were mechanically lysed and then incubated for 30 min at 4°C with rotation. Lysates were precleared with normal rabbit IgG (2 μg) bound protein G agarose for 1 hr at 4°C and then clarified by centrifugation. Clarified lysates were quantified by BCA (Pierce) and normalized. Input samples were reserved and either 2 or 5 mg of lysate was immunoprecipitated with either 2 or 3 μg of rabbit α-Cx45 (Alomone) ON at 4°C. Immune-protein complexes were captured by 2 hr 4°C incubation with protein G agarose (Pierce), washed 5x with IP wash buffer (IP lysis buffer excluding sodium deoxycholate). Immunoprecipitated proteins were eluted using 35–50 μL 3x SDS sample buffer. Equal volumes of each sample were analyzed by western blot and to avoid cross reactivity with the IP antibody a light chain specific secondary antibody (Jackson) or Veriblot (Abcam) were utilized for detection. Quantification was done using NIH ImageJ software from 3 independent replicates.
Trease A.J., Capuccino J.M., Contreras J.E., Harris A.L, & Sorgen P.L. (2017). Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization. Journal of molecular and cellular cardiology, 111, 69-80.
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3

Immunoblotting Protein Analysis Protocol

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Unless otherwise stated, general lab reagents were purchased from Sigma Aldrich and were of the highest quality available. The following primary antibodies were used for immunoblotting at 1/1000 dilution in 5% (w/w) BSA overnight at 4°C: anti-RelA (sc-372, Santa Cruz), anti-HA (Merck, HA-7), anti phospho-histone H2AX (#2577, Cell Signalling), anti-PAK4 (#3242, Cell Signaling Technologies), anti-Karyopherin-β1 H-7 (sc-137016, Santa Cruz). Secondary anti-rabbit IgG (7074S, Cell Signalling Technology) was used at 1/5000 dilution following membrane incubation with VeriBlot (1/400 dilution, ab131366 Abcam).
Campbell A.E., Ferraz Franco C., Su L.I., Corbin E.K., Perkins S., Kalyuzhnyy A., Jones A.R., Brownridge P.J., Perkins N.D, & Eyers C.E. (2021). Temporal modulation of the NF-κB RelA network in response to different types of DNA damage. Biochemical Journal, 478(3), 533-551.
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4

Whole Cell Protein Lysis and Western Blotting

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Whole cell protein lysates were prepared using either RSB100 (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 2.5 mM MgCl2, 0.5% NP-40, 0.5% Triton X-100) or TOPEX+ buffer (Riising et al., 2014 (link)) (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 0.5% Triton X-100, 1% SDS, 33.3 U/mL Benzonase) freshly supplemented with protease inhibitors (Roche). Samples were denatured by the addition of NuPAGE Loading Buffer (Invitrogen) and NuPAGE Sample Reducing Agent (Invitrogen) before boiling at 95°C for 10 min. SDS-PAGE was carried out on either NuPAGE 4%–12% Bis-Tris or 3%–8% Tris-Acetate gels (Invitrogen). Western blotting analysis was carried out according to standard protocols with the antibodies listed in the key resources table and HRP conjugated secondary antibodies (Agilent) or Veriblot (Abcam). Bands were visualized by Super Signal West Fempto chemiluminescent ECL (Thermo) and captured using an Amersham Imager 600 or ImageQuant 800 imaging systems (GE Healthcare). Images were processed and quantified using ImageJ (v1.51) (Schneider et al., 2012 (link)).
Garland W., Müller I., Wu M., Schmid M., Imamura K., Rib L., Sandelin A., Helin K, & Jensen T.H. (2022). Chromatin modifier HUSH co-operates with RNA decay factor NEXT to restrict transposable element expression. Molecular cell, 82(9), 1691-1707.e8.
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5

Western Blot Analysis of Influenza Proteins

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Cell lysates obtained as described above were mixed with SDS electrophoresis sample buffer containing 90 mM DDT, boiled and subjected to electrophoresis in 4–12% gradient Novex NuPAGE SDS-PAGE Gels (Invitrogen). Resolved proteins were transferred to Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore, Etobicoke, ON, Canada), and membranes were blocked in 5% skim milk and probed with various antibodies. Primary antibodies were monoclonal α-NS1 [42 (link)], monoclonal α-NP [44 (link)] (gift from Dr. Mingyi Li, National Microbiology Labs), monoclonal α-M1 (Thermo Fisher, MA1-80736), polyclonal α-M2 (Thermo Fisher, PA5-32233), monoclonal α-beta-actin (Cell Signalling, 3700S, Danvers, MA, USA), α-NUMA1 (Bethyl Laboratory, A301-510A, Montgomery, TX, USA), α-PRPF19 (Bethyl Laboratory, A300-101A) and/or α-UTP6 (Thermo Fisher, PA5-21716). Primary antibodies were detected with HRP-linked polyclonal α-mouse (Cell Signalling, 7076S), polyclonal α-rabbit (Cell Signalling, 7074S) or monoclonal VeriBlot™ (Abcam, Ab131366, Toronto, ON, Canada) secondary antibodies and HRP signals were detected using enhanced chemiluminescence (ECL) reagent (prepared in house). Images were taken with an Alpha Innotech Fluor Chem® Q Imaging System and minimally processed by Adobe® Photoshop® software (San Jose, CA, USA).
Rahim M.N., Klewes L., Zahedi-Amiri A., Mai S, & Coombs K.M. (2018). Global Interactomics Connect Nuclear Mitotic Apparatus Protein NUMA1 to Influenza Virus Maturation. Viruses, 10(12), 731.
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6

Whole Cell Protein Lysis and Western Blotting

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Whole cell protein lysates were prepared using either RSB100 (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 2.5 mM MgCl2, 0.5% NP-40, 0.5% Triton X-100) or TOPEX+ buffer (Riising et al., 2014 (link)) (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 0.5% Triton X-100, 1% SDS, 33.3 U/mL Benzonase) freshly supplemented with protease inhibitors (Roche). Samples were denatured by the addition of NuPAGE Loading Buffer (Invitrogen) and NuPAGE Sample Reducing Agent (Invitrogen) before boiling at 95°C for 10 min. SDS-PAGE was carried out on either NuPAGE 4%–12% Bis-Tris or 3%–8% Tris-Acetate gels (Invitrogen). Western blotting analysis was carried out according to standard protocols with the antibodies listed in the key resources table and HRP conjugated secondary antibodies (Agilent) or Veriblot (Abcam). Bands were visualized by Super Signal West Fempto chemiluminescent ECL (Thermo) and captured using an Amersham Imager 600 or ImageQuant 800 imaging systems (GE Healthcare). Images were processed and quantified using ImageJ (v1.51) (Schneider et al., 2012 (link)).
Garland W., Müller I., Wu M., Schmid M., Imamura K., Rib L., Sandelin A., Helin K, & Jensen T.H. (2022). Chromatin modifier HUSH co-operates with RNA decay factor NEXT to restrict transposable element expression. Molecular cell, 82(9), 1691-1707.e8.
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7

Immunoprecipitation of CD98hc Protein

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Subconfluent cells in 10‐cm dishes (Corning) were washed with PBS and lysed on ice for 30 minutes in 3 mL lysis buffer containing 1% Nonidet P‐40 and protease inhibitor cocktail (Nacalai Tesque). Resulting extracts were centrifuged at 19 000 x g for 10 minutes at 4°C, and the supernatants were precleared with protein G Sepharose for 1 hour. Lysates were subjected to immunoprecipitation with antirat CD98hc (B3) or antihuman CD98hc (HBJ127) mouse mAb for 6 hours at room temperature, followed by the addition of protein G Sepharose and further incubation for 2 hours. Bead‐bound proteins were washed three times with 1% (v/v) Nonidet P‐40 in PBS, suspended in 1× SDS‐PAGE loading buffer, and boiled for 5 minutes, and the beads were then isolated by centrifugation. For immunoblotting analysis, proteins were solubilized in loading buffer, subjected to SDS‐PAGE (8% gels) and transferred to Immobilon‐P (Millipore), and exposed to rabbit anti‐GFP pAb. Immune complexes were detected by HRP‐conjugated VeriBlot (ab131366; Abcam, Cambridge, UK) and Chemi‐Lumi One Super (Nacalai Tesque).
Ueda S., Hayashi H., Miyamoto T., Abe S., Hirai K., Matsukura K., Yagi H., Hara Y., Yoshida K., Okazaki S., Tamura M., Abe Y., Agatsuma T., Niwa S., Masuko K, & Masuko T. (2019). Anti‐tumor effects of mAb against l‐type amino acid transporter 1 (LAT1) bound to human and monkey LAT1 with dual avidity modes. Cancer Science, 110(2), 674-685.
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8

Western Blot Analysis of GABA Receptor Subunits

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Lysates and beads were boiled in 2x SDS-sample buffer (8% SDS, 40% glycerol, 0.25 M Tris pH 6.8, 20% β-mercaptoethanol) for 5 minutes, separated on a 10% SDS-PAGE, and blotted onto PVDF membranes (Roche). Membranes were blocked with 5% skim milk/PBST. Primary antibodies were diluted in the blocking buffer and applied over night at 4°C, followed by 3 washes with PBST and 1 hour incubation with appropriate secondary antibodies. HRP signals were detected using Western Lightning chemiluminescent substrates (Perkin Elmer, USA) with a luminescent image analyser (ImageQuant LAS4000, GE Healthcare).
Primary antibodies: αGABAAR α1 (mouse, 75–136, NeuroMab), αFlag (mouse, F1804, Sigma), αFlag-HRP (mouse, A8592, Sigma), αGFP (chicken, ab13970, Abcam), αGST (mouse, 75–148, NeuroMab), and αHA (rabbit, H6928, Sigma).
Secondary antibodies: αMouse-HRP (115-035-003, Dianova), αMouse-native-IgG (Veriblot, 131368, Abcam), αRabbit-HRP (111-035-003, Dianova), and αChicken-HRP (ab6753, Abcam).
Schmerl B., Gimber N., Kuropka B., Stumpf A., Rentsch J., Kunde S.A., von Sivers J., Ewers H., Schmitz D., Freund C., Schmoranzer J., Rademacher N, & Shoichet S.A. (2022). The synaptic scaffold protein MPP2 interacts with GABAA receptors at the periphery of the postsynaptic density of glutamatergic synapses. PLoS Biology, 20(3), e3001503.
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9

Identifying CatSper Protein Interactions

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Solubilized testis microsome and round spermatids were subjected to coIP. Solubilized proteins in lysis buffer were mixed with SureBeads Protein A Magnetic Beads (BioRad) conjugated with either 1 μg of rabbit polyclonal anti-CatSper1, CatSperδ, or CatSperτ(α-CST-482), or 0.5 μL of rabbit monoclonal anti-DYKDDDDK (clone D6W5B, CST). After incubation for overnight at 4°C, the resins were washed with lysis buffer and eluted immunocomplexes were eluted with 2X LDS buffer supplemented with 50 μM DTT by boiling at 75°C for 10 min. The elutes were subjected to SDS-PAGE and immunoblotting. Primary antibodies for immunoblotting were: rabbit polyclonal anti-mouse CatSper1, CatSper2, CatSper3, CatSperβ, CatSperδ, CatSperɛ, and CatSperτ at 1 μg/mL, and rabbit monoclonal anti-DYKDDDDK (1:2,000; clone D6W5B, CST). For secondary antibodies, VeriBlot (1:200-1:500; Abcam) and HRP-conjugated goat anti-mouse or rabbit IgG (1:10,000; Jackson ImmunoResearch) were used.
Hwang J.Y., Wang H., Lu Y., Ikawa M, & Chung J.J. (2022). C2cd6-encoded CatSperτ targets sperm calcium channel to Ca2+ signaling domains in the flagellar membrane. Cell reports, 38(3), 110226.
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10

Monoclonal Antibody-Based SerpinE2 Detection

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A monoclonal serpinE2 antibody which recognizes rat and mouse serpinE2 was used for westerns (clone 4B3, production described in [17 (link)]). Recombinant tPA was purchased and bound to recombinant serpinE2 as described previously [41 (link)]. Human recombinant serpinE2 and tPA were purchased from R&D. Anti-human SerpinE2 Ab (MAB2980) was from R&D systems. The secondary antibody for Western blotting was anti-mouse IgG (LNA931V/AG, GE Healthcare UK), or Veriblot (ab131368, Abcam) for immunoprecipitation experiments. The anti-mouse antibodies for the FACS analyses were all from BioLegend: CD11b (APC-CD11b, cl.M1/70), Ly6C (FITC-LY6C, cl.HK1.4), Ly6G (Percp-Cy5.5-Ly6G, cl.IA8), CD206 (FITC-CD206, cl.C068C2), MHCII (Percp-Cy5.5-I-A/I-E, cl.M5/114.15.2), CD11c (Percp-Cy5.5-CD11c, cl.N418), CD86 (Alexa-fluor 488 CD86, cl.GL-1), CD45 (BV-785); F4/80 (PE, was from eBioscience). Liposomes were purchased from www.ClodronateLiposomes.com.
Smirnova T., Bonapace L., MacDonald G., Kondo S., Wyckoff J., Ebersbach H., Fayard B., Doelemeyer A., Coissieux M.M., Heideman M.R., Bentires-Alj M, & Hynes N.E. (2016). Serpin E2 promotes breast cancer metastasis by remodeling the tumor matrix and polarizing tumor associated macrophages. Oncotarget, 7(50), 82289-82304.
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