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Hrp conjugated actin

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HRP-conjugated actin is a protein that is commonly used in various laboratory techniques for the detection and visualization of actin, a major structural component of the cytoskeleton in eukaryotic cells. It is covalently linked to the enzyme horseradish peroxidase (HRP), which is used as a reporter molecule to generate a colorimetric or chemiluminescent signal when exposed to an appropriate substrate.

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Lab products found in correlation

Bca protein assay, by Thermo Fisher Scientific (4 mentions) Ab4469, by Abcam (2 mentions) Suz12, by Cell Signaling Technology (2 mentions) Nupage novex 4 12 bis tris protein gel, by Thermo Fisher Scientific (2 mentions) 0.45 μm nitrocellulose membrane, by Bio-Rad (2 mentions) Hrp conjugated total h3, by Cell Signaling Technology (2 mentions) Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions) H3k27me3, by Cell Signaling Technology (2 mentions) Ripa buffer, by Cell Signaling Technology (2 mentions) H3k27ac, by Abcam (2 mentions) Cd63 sc 5275, by Santa Cruz Biotechnology (2 mentions) Vinculin, by Merck Group (2 mentions) Nsmase2a sc 166637, by Santa Cruz Biotechnology (2 mentions) Cgas sab2100310, by Merck Group (2 mentions) Sting, by R&D Systems (2 mentions) Calnexin, by Abcam (2 mentions) Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (2 mentions) Lamin a c, by Cell Signaling Technology (2 mentions) Gapdh, by Cell Signaling Technology (2 mentions) Rvfv nucleoprotein, by BEI Resources (2 mentions)

8 protocols using hrp conjugated actin

1

Western Blot Analysis of VEEV Proteins

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Whole cell lysates were separated on a 4–20% Tris–Glycine Gel at 100 V and transferred to a polyvinyl difluoride (PVDF) membrane using the iBlot gel transfer system (Invitrogen). The membranes were blocked in 1% dry milk in PBS-Tween 20 or 2% BSA in PBS-Tween 20 at room temperature. Primary antibodies to VEEV Capsid (BEI Resources, NR 9403), VEEV Glycoprotein (BEI Resources, NR 9404), p-ERK 1/2 (Thr 202/Tyr 204) (Cell Signaling Technology, Catalogue no. 4370), ERK 1/2 Antibody (Cell Signaling Technology, Catalogue no. 9102), p-p90RSK (Ser380) (Cell Signaling Technology, Catalogue no. 11,989) and HRP-conjugated actin (Abcam, Catalogue no. ab49900) were used according to manufacturer׳s instructions. The blots were incubated with primary antibody overnight at 4 °C. Following 2 washes with PBS-Tween 20 the blots were then incubated with respective secondary HRP-coupled antibody for 2 h. After 3 washes with PBS-Tween 20 and 1 wash with PBS, the membranes were visualized by chemiluminescence using SuperSignal West Femto Maximum Sensitivity Substrate Kit (ThermoScientific) on a BIO- RAD Molecular Imager ChemiDoc XRS system (BIO-RAD).
Voss K., Amaya M., Mueller C., Roberts B., Kehn-Hall K., Bailey C., Petricoin E I.I.I, & Narayanan A. (2014). Inhibition of host extracellular signal-regulated kinase (ERK) activation decreases new world alphavirus multiplication in infected cells. Virology, 468, 490-503.
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2

Protein Lysate Production and Western Blotting

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Protein lysates production and western blotting were performed as described (Baer et al., 2012 (link), 2016b (link)). Blots were probed with anti-cyclin D1 (Cell signaling Cat#2978) anti-cyclin E2 (Cell Signaling Cat#4132), anti-cyclin A2 (Cell Signaling Cat#4656), anti-VEEV capsid (BEI Resources, NR 9403), and HRP-conjugated actin (catalog number ab49900-100, Abcam) antibodies.
Lundberg L., Fontenot J., Lin S.C., Pinkham C., Carey B.D., Campbell C.E, & Kehn-Hall K. (2018). Venezuelan Equine Encephalitis Virus Capsid Implicated in Infection-Induced Cell Cycle Delay in vitro. Frontiers in Microbiology, 9, 3126.
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3

Western Blot Analysis of Chromatin Regulators

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Cell lysates were prepared in RIPA buffer (#9806, Cell Signaling) supplemented with Halt protease and phosphatase inhibitor cocktail (#78440, Thermo Scientific). Equal amounts of protein, as measured by BCA protein assay (#23225, Thermo Scientific), were resolved in NuPAGE® Novex® 4–12% Bis-Tris Protein Gels (#NP0321BOX, Life Technologies) and transferred electrophoretically onto a Nitrocellulose 0.45 μm membrane (#162-0115, BioRad). Membranes were blocked for 1 hour at room temperature in 5% BSA in TBST before being incubated overnight at 4°C with the primary antibodies diluted at 1:1000 in 5% BSA in TBST. NF1 (#sc-67, Santa Cruz, 1:500), EZH1 (#ab13665, Abcam, 1:1000), EZH2 (#5246, Cell signaling,1:1000), SUZ12 (#3737, Cell Signaling, 1:1000), EED (#ab4469, Abcam, 1:1000), H3K27me3 (#9733, Cell Signaling, 1:1000), H3K27Ac (#ab4729, Abcam, 1:1000), HRP-conjugated Total H3 (#12648, Cell Signaling, 1:1000), and HRP-conjugated Actin(#ab49900, Abcam, 1:5000) antibodies were used.
Lee W., Teckie S., Wiesner T., Ran L., Prieto Granada C.N., Lin M., Zhu S., Cao Z., Liang Y., Sboner A., Tap W.D., Fletcher J.A., Huberman K.H., Qin L.X., Viale A., Singer S., Zheng D., Berger M.F., Chen Y., Antonescu C.R, & Chi P. (2014). PRC2 is recurrently inactivated through EED or SUZ12 loss in malignant peripheral nerve sheath tumors. Nature genetics, 46(11), 1227-1232.
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4

Comprehensive Western Blot Analysis of Exosome Markers

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Cells were lysed using RIPA buffer in the presence of protease and phosphatase inhibitors. The centrifuged supernatants were subjected to SDS-PAGE (Criterion™ TGX™) and immunoblotting. Transfer to PVDF membranes was carried out using Trans-Blot Turbo™ Transfer System®. The blots were blocked with 2.5% BSA in PBS containing 0.1% Tween20 before incubating with the following primary antibodies: CD81 (sc-9158, Santa Cruz), CD63 (sc-5275, Santa Cruz), calnexin (ab-22595, Abcam), nSmase2a (sc-166637, Santa Cruz), cGAS (SAB2100310, Sigma-Aldrich), STING (AF6516, R&D), vinculin (V9131, Sigma), HRP-conjugated actin (ab-49906, Abcam). Corresponding peroxidase-conjugated secondary antibodies were used (Jackson ImmunoResearch). Membranes were developed using Super Signal West Dura extended duration substrate (Thermo Scientific, 34076). Original blots are shown in Supplementary Figure 7.
Nandakumar R., Tschismarov R., Meissner F., Prabakaran T., Krissanaprasit A., Farahani E., Zhang B.C., Assil S., Martin A., Bertrams W., Holm C.K., Ablasser A., Klause T., Thomsen M.K., Schmeck B., Howard K.A., Henry T., Gothelf K.V., Decker T, & Paludan S.R. (2019). Intracellular bacteria engage a STING-TBK1-MVB12b pathway to enable paracrine cGAS-STING signaling. Nature microbiology, 4(4), 701-713.
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5

Western Blot Analysis of Protein Lysates

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Protein lysates were collected and analyzed by western blot as previously described24 (link). In brief, primary antibodies against GAPDH (Cell Signaling, 5174), Lamin A/C (Cell Signaling, 4777), p-Akt1 Ser 473 (Cell Signaling, 4060), total Akt1 (Cell Signaling, 4691), IFIT2 (Abcam - ab113112), Viperin (Cell Signaling -13996 S), Actinin 2 (Thermo Fisher Scientific - PA5-27863), Alpha-Pix (Cell Signaling -4753 S), RVFV Nucleoprotein (BEI Resources, NR-43188), or HRP-conjugated actin (Abcam, ab49900) were diluted in 3% milk solution per the manufacturer’s recommended dilutions followed by the addition of the appropriate secondary antibody. Cropped, representative images are shown in main figures, but uncropped originals can be found in the Supplemental Figure 8.
Pinkham C., Dahal B., de la Fuente C.L., Bracci N., Beitzel B., Lindquist M., Garrison A., Schmaljohn C., Palacios G., Narayanan A., Campbell C.E, & Kehn-Hall K. (2017). Alterations in the host transcriptome in vitro following Rift Valley fever virus infection. Scientific Reports, 7, 14385.
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6

Comprehensive Western Blot Analysis of Exosome Markers

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Cells were lysed using RIPA buffer in the presence of protease and phosphatase inhibitors. The centrifuged supernatants were subjected to SDS-PAGE (Criterion™ TGX™) and immunoblotting. Transfer to PVDF membranes was carried out using Trans-Blot Turbo™ Transfer System®. The blots were blocked with 2.5% BSA in PBS containing 0.1% Tween20 before incubating with the following primary antibodies: CD81 (sc-9158, Santa Cruz), CD63 (sc-5275, Santa Cruz), calnexin (ab-22595, Abcam), nSmase2a (sc-166637, Santa Cruz), cGAS (SAB2100310, Sigma-Aldrich), STING (AF6516, R&D), vinculin (V9131, Sigma), HRP-conjugated actin (ab-49906, Abcam). Corresponding peroxidase-conjugated secondary antibodies were used (Jackson ImmunoResearch). Membranes were developed using Super Signal West Dura extended duration substrate (Thermo Scientific, 34076). Original blots are shown in Supplementary Figure 7.
Nandakumar R., Tschismarov R., Meissner F., Prabakaran T., Krissanaprasit A., Farahani E., Zhang B.C., Assil S., Martin A., Bertrams W., Holm C.K., Ablasser A., Klause T., Thomsen M.K., Schmeck B., Howard K.A., Henry T., Gothelf K.V., Decker T, & Paludan S.R. (2019). Intracellular bacteria engage a STING-TBK1-MVB12b pathway to enable paracrine cGAS-STING signaling. Nature microbiology, 4(4), 701-713.
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7

Western Blot Analysis of Chromatin Regulators

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Cell lysates were prepared in RIPA buffer (#9806, Cell Signaling) supplemented with Halt protease and phosphatase inhibitor cocktail (#78440, Thermo Scientific). Equal amounts of protein, as measured by BCA protein assay (#23225, Thermo Scientific), were resolved in NuPAGE® Novex® 4–12% Bis-Tris Protein Gels (#NP0321BOX, Life Technologies) and transferred electrophoretically onto a Nitrocellulose 0.45 μm membrane (#162-0115, BioRad). Membranes were blocked for 1 hour at room temperature in 5% BSA in TBST before being incubated overnight at 4°C with the primary antibodies diluted at 1:1000 in 5% BSA in TBST. NF1 (#sc-67, Santa Cruz, 1:500), EZH1 (#ab13665, Abcam, 1:1000), EZH2 (#5246, Cell signaling,1:1000), SUZ12 (#3737, Cell Signaling, 1:1000), EED (#ab4469, Abcam, 1:1000), H3K27me3 (#9733, Cell Signaling, 1:1000), H3K27Ac (#ab4729, Abcam, 1:1000), HRP-conjugated Total H3 (#12648, Cell Signaling, 1:1000), and HRP-conjugated Actin(#ab49900, Abcam, 1:5000) antibodies were used.
Lee W., Teckie S., Wiesner T., Ran L., Prieto Granada C.N., Lin M., Zhu S., Cao Z., Liang Y., Sboner A., Tap W.D., Fletcher J.A., Huberman K.H., Qin L.X., Viale A., Singer S., Zheng D., Berger M.F., Chen Y., Antonescu C.R, & Chi P. (2014). PRC2 is recurrently inactivated through EED or SUZ12 loss in malignant peripheral nerve sheath tumors. Nature genetics, 46(11), 1227-1232.
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8

Western Blot Analysis of Protein Lysates

Cited 1 time
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Protein lysates were collected and analyzed by western blot, or cellular fractionation was performed and analyzed by western blot, as previously described (Austin et al., 2012 (link)). In brief, primary antibodies against p-STAT3 Y705 (Cell Signaling, 9145S), total STAT3 (Cell Signaling, 12640S), cleaved Caspase-3 (Cell Signaling, 9661S), Flag (Sigma, F1804), RVFV Nucleoprotein (BEI Resources, NR-43188), Lamin A/C (Cell Signaling, 4777S), GAPDH (Cell Signaling, 5174S), or HRP-conjugated actin (Abcam, ab49900) were diluted in 3% milk solution according to the manufacturer’s recommended dilutions followed by the addition of the appropriate secondary antibody.
Pinkham C., An S., Lundberg L., Bansal N., Benedict A., Narayanan A, & Kehn-Hall K. (2016). The Role of Signal Transducer and Activator of Transcription 3 in Rift Valley Fever Virus Infection. Virology, 496, 175-185.
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