The FTA was performed as described previously.41 (link),42 (link) Briefly, HeLa cells stably expressing Exon1Htt103Q-HT were maintained without doxycycline for several passages to stabilize aggregate number. Cells were then plated and transfected with Lipofectamine 2000 per manufacturer’s instructions with tdTomato, tdTomato-ALFYC or tdTomato-ALFYC E3471K. 24 hours after transfection, a subset of wells transfected with tdTomato were treated with dox to inhibit Exon1Htt103Q-HT expression. All samples were collected then lysed in PBS + 1% Triton-X100 + 8M Urea 96 hours post-transfection. Lysates were immediately pelleted at 14000 rpm. Lysates were quantified using a DC protein assay kit (Bio-Rad). 120 μg and 30 μg of protein were then slotted onto a cellulose acetate membrane (20 μM) using a Biodot (Biorad) and probed with anti-Halotag antibody (Promega). 20 μg of protein was also run on the 4-12% Bis Tris gels (ThermoFisher) and probed with anti-Halotag and anti-Vinculin (ThermoFisher). Densitometry was performed on ImageJ.
Anti halotag antibody
Manufactured by Promega
The Anti-HaloTag antibody is a laboratory reagent used to detect the presence of the HaloTag protein in biological samples. The HaloTag is a protein tag that can be fused to target proteins, allowing for their detection and manipulation. The Anti-HaloTag antibody binds to the HaloTag and enables visualization or immunoprecipitation of the tagged proteins.
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Lab products found in correlation
Anti β actin antibody, by Merck Group (3 mentions)
Sc 166025, by Santa Cruz Biotechnology (2 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Seamless cloning kit, by Beyotime (1 mentions)
Anti gr, by Santa Cruz Biotechnology (1 mentions)
Anti tfiib, by Santa Cruz Biotechnology (1 mentions)
Hiseq sequencing platform, by Illumina (1 mentions)
Tnt sp6 coupled wheat germ extract system, by Promega (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
12 mm glass coverslips, by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Tmr conjugated halo ligand, by Promega (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Anti v5 antibody, by Thermo Fisher Scientific (1 mentions)
Lds sample buffer, by Thermo Fisher Scientific (1 mentions)
L1170, by Promega (1 mentions)
Phalloidin 647, by Thermo Fisher Scientific (1 mentions)
16 protocols using anti halotag antibody
1
Aggregate Formation Monitoring in HeLa Cells
2
Quantifying SOX18 Protein Levels
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Western blotting was used to assess the level of endogenous SOX18 protein in HeLa cells and HUVECs (Supplementary Figure S2A ), and to compare the expression level and nuclear concentration of overexpressed HALO-SOX18 and HALO-SOX18RaOp protein in HeLa cells (Supplementary Figure S2C ). Cells were seeded, and either transfected (using 1 μg of expression plasmid to 3 μl of X-tremeGENE 9) or left untransfected, and harvested for either whole cell lysates or nuclear extracts before subjecting to SDS-PAGE and Western Blotting with a human anti-SOX18 antibody (sc166025 from Santa Cruz), anti-HaloTag antibody (G9281 from Promega), or housekeeping control anti-β-actin antibody (A5441 from Sigma).
3
Cell-Free Expression of Wheat Transcription Factor IRR
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Details of wheat cell-free reaction have been described previously [39 (link)]. Cell-free protein expression was performed using the TNT SP6 Wheat Germ Master Mix Kit (Promega, Madison, WI). The primers for the irr gene are as follows: irr forward, 5΄-CCATATGATGCATTCTTCACATACCCA-3΄; irr reverse, 5΄-CTCTAGATCAGCGGGCCTGACGGCG-3΄. The polymerase chain reactions (PCRs) were incubated at 95°C for 5 min. Then 35 cycles were performed as follows: 30 s at 95°C, 40 s at 57°C, and 7 min at 72°C. The amplified PCR product was subcloned into a pDAP-Halo-Kan vector (Zoobio, Nanjing, China) using the Seamless cloning kit (Beyotime, Shanghai, China), and the recombinant plasmid was used as a transcription template. The protein expression volume was 50 μL, comprising 30 μL Wheat Germ Master Mix, 1 μg pDAP-Halo-Kan-irr plasmid, and nuclease-free water to make 50 μL; this was incubated at 25°C for 2 h. The expressed protein was confirmed using western blot analysis and an anti-Halo tag antibody (Promega, Madison, WI).
4
Antibody Profiling for Transcriptional Regulation
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The following antibodies were used: anti-GR and anti-GRIP1 (BD Biosciences); anti-GR (rabbit, H300 Santa Cruz Biotechnology); anti-GR (rabbit, Novus Biological); anti-Merm1 (WBSCR22) (mouse, Abcam); anti-Merm1 (WBSCR22) (rabbit, Source Bioscience); anti-histone H3 acetyl (rabbit, Millipore); anti-histone H3 lysine 4 trimethyl (rabbit, Millipore) and anti-histone H3 lysine 79 dimethyl (rabbit, Millipore); anti-pan-methyl histone H3 (Lys-9) antibody (rabbit, Cell Signaling); anti-TFIIB (rabbit, Santa Cruz Biotechnology); horseradish peroxidase-conjugated anti-mouse and anti-rabbit from GE Healthcare. Anti-Halo-tag antibody (Promega). Mouse IgG and rabbit IgG were from Millipore. Fluorophore-conjugated (Alexa Fluor 546 and 488) anti-mouse and anti-rabbit antibodies were from Invitrogen.
5
Genomic DNA Binding Assay for OsMYB80 Transcription Factor
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The DAP-seq analysis was performed according to the protocol described by Bartlett et�al. (2017) (link). Briefly, 5 μg HHZ genomic DNA was sheared into ∼200-bp fragments and then ligated with the Illumina-based sequencing adaptors to form a DNA library. The library was examined for adaptor-ligation frequency by quantitative PCR (qPCR) before applied to DAP-seq assay.
OsMYB80 ORF fused to the Halo affinity tag was expressed in vitro using TNT� SP6 Coupled Wheat Germ Extract System (Promega, Fitchburg, WI, USA). The HaloTag-OsMYB80 protein was then purified from nonspecific proteins in the expression system using the magnetic HaloTag ligand (chloroalkane) beads (Promega) and verified by Western blotting with the anti-HaloTag antibody (Promega). The purified protein was incubated with 500 ng adaptor-ligated genomic DNA library at 30�C for 2 h before washing away the unbound DNA fragments.
The samples were heated at 98�C for 10 min to release the OsMYB80-bound DNA, and the recovered DNA was then PCR-amplified with the indexed TruSeq primers (Illumina, San Diego, CA, USA). Indexed DNA samples were subsequently combined and size-selected to remove the residual adaptor dimers. Purified DNA libraries were then sequenced using the Illumina HiSeq sequencing platform.
OsMYB80 ORF fused to the Halo affinity tag was expressed in vitro using TNT� SP6 Coupled Wheat Germ Extract System (Promega, Fitchburg, WI, USA). The HaloTag-OsMYB80 protein was then purified from nonspecific proteins in the expression system using the magnetic HaloTag ligand (chloroalkane) beads (Promega) and verified by Western blotting with the anti-HaloTag antibody (Promega). The purified protein was incubated with 500 ng adaptor-ligated genomic DNA library at 30�C for 2 h before washing away the unbound DNA fragments.
The samples were heated at 98�C for 10 min to release the OsMYB80-bound DNA, and the recovered DNA was then PCR-amplified with the indexed TruSeq primers (Illumina, San Diego, CA, USA). Indexed DNA samples were subsequently combined and size-selected to remove the residual adaptor dimers. Purified DNA libraries were then sequenced using the Illumina HiSeq sequencing platform.
6
Confocal Imaging of Lamin A Mutants in U2OS Cells
7
HaloTag-LC3 Protein Expression Monitoring
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Halo-LC3 stable expression cells were generated by viral transduction. The pMRX-IP-HaloTag7-LC3 was used for gene transduction. Cells were incubated in DMEM with 100 nM TMR-conjugated Halo ligand (Promega; catalog no.: G8251) for 1 h. Western blotting with an anti-HaloTag antibody (Promega; catalog no.: G9211) was performed to assess the amount of Halo-LC3 and its degradation products. pMRX-IP-HaloTag7-LC3 was a gift from Noboru Mizushima (Addgene plasmid; catalog no.: 184899; http://n2t.net/addgene:184899 ; Research Resource Identifer: Addgene_184899) (17 ).
8
Western Blot Analysis of Cellular Proteins
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HepG2 or Huh7 cells were washed twice with ice‐cold PBS and ruptured with RIPA buffer containing protease inhibitor (Roche, #1697498). Cell lysates were resolved by SDS‐polyacrylamide gel electrophoresis (PAGE) and then transferred onto PVDF membranes. The membranes were blocked for 1 hour with 5% non‐fat milk in TBS‐T and incubated with anti‐HaloTag antibody (Promega, #G928A), anti‐HMGA2 antibody (Active Motif, #61041), anti‐V5 antibody (Invitrogen, #1305726), anti‐nucleoporin antibody (BD Biosciences, #610498), or anti‐β‐actin antibody (Sigma, #A5441) at 4°C overnight. Membranes were washed for 20 minutes with TBS‐T, probed with appropriate HRP‐conjugated secondary antibodies, and detected with the ECL Plus chemi‐luminescence assay kit (GE Healthcare).
9
Halo-tagged ASPP Family CTD Pulldown
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One hundred micrograms of purified Halo or Halo-tagged ASPP family CTDs (ASPP1: 887–1090, ASPP2:925–1128) were covalently immobilized on 25 μl magnetic Halo beads slurry in pulldown buffer (50 mM HEPES pH 7.5, 200 mM NaCl, 0.5 mM TCEP, 0.1% Surfactant P-20) overnight at 4 °C. The following day, myc-tagged NS5A wild-type and mutants of the indicated HCV strains or HCV core protein were produced by in vitro translation (Promega; L1170) from pcDNA3.1(+) plasmids. Reactions were incubated for 90 min at 30 °C. DNA and RNA was digested by using benzonase nuclease for 30 min at 4 °C and reactions were cleared by centrifugation for 10 min at 13 000 g to remove aggregates. Loaded beads were washed three times with pull-down buffer to remove unbound Halo proteins and incubated with 20 μl in vitro translated protein and 10 μM dissolved peptide, if indicated. Following rotation for 3 h at 4 °C, beads were washed four times with ice-cold pulldown buffer and bound proteins were eluted in 1× LDS sample buffer (Thermo Fisher) supplemented with 0.5 mM DTT for 10 min at 70 °C. Input and pulldown samples were analysed by Western blot using anti-myc tag antibody (Merck Millipore) and anti-Halo tag antibody (Promega).
10
Dissecting EGF Signaling Regulation
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