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Anti tap antibody

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The Anti-TAP antibody is a laboratory reagent used for protein purification and identification. It binds to the Tandem Affinity Purification (TAP) tag, which is a protein tag commonly used to facilitate the isolation of protein complexes from cell lysates. The antibody can be used in various applications, such as immunoprecipitation and Western blotting, to detect and purify TAP-tagged proteins.

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Lab products found in correlation

Anti his antibody, by Merck Group (2 mentions) Anti phosphot172 ampk antibody, by Cell Signaling Technology (2 mentions) β actin clone c4, by MP Biomedicals (1 mentions) Anti gluc antibody, by New England Biolabs (1 mentions) Anti flag antibody, by Merck Group (1 mentions) Rotor gene q, by Qiagen (1 mentions) Hrp linked goat anti rabbit antibody, by Abcam (1 mentions) Anti ha antibody, by Roche (1 mentions) Protein assay, by Bio-Rad (1 mentions) Typhoon trio variable mode imager, by GE Healthcare (1 mentions) Imagequant tl software package, by GE Healthcare (1 mentions) Calmodulin sepharose beads, by GE Healthcare (1 mentions) Igg sepharose fast flow beads, by GE Healthcare (1 mentions)

Spelling variants of this product

Anti-TAP (9 citations) Anti-TAP antibody (CAB1001) (5 citations) Anti-TAP tag rabbit polyclonal antibody (2 citations)

8 protocols using anti tap antibody

1

Endogenous Protein Detection by Western Blot

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Western blot detection of endogenous proteins was performed using the following antibodies: p73 (Abcam, ref: ab215038), E2F4 (Santa Cruz Biotechnology, ref. sc-398543X), E2F5 (Genetex, ref. GTX129491), DP1 (Abcam, ref. ab124678), p130 (Cell Signaling, ref. 13610S) β-actin (clone C4, MP Biomedicals), and GAPDH (6C5, ref. sc-32233, Santa Cruz). Detection of tagged constructs was done using: HA-peroxidase antibody (Roche, ref: 12013819001), anti-TAP antibody (Thermofisher Scientific, ref. CAB1001), anti-Flag antibody (Sigma, ref. F3165) antibody, and anti-Gluc antibody (New England Biolabs, ref. E8023).
Taverniti V., Krynska H., Venuti A., Straub M.L., Sirand C., Lohmann E., Romero-Medina M.C., Moro S., Robitaille A., Negroni L., Martinez-Zapien D., Masson M., Tommasino M, & Zanier K. (2023). The E2F4/p130 Repressor Complex Cooperates with Oncogenic ΔNp73α To Inhibit Gene Expression in Human Papillomavirus 38 E6/E7-Transformed Keratinocytes and in Cancer Cells. mSphere, 8(2), e00056-23.
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2

Quantifying Snf1/AMPK Phosphorylation

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To assess the Snf1/AMPK phosphorylation state, cells were quenched using 6% TCA (Merck Life Science, Milan, Italy). Then, the cells were lysed in UREA buffer (6 M UREA, 1% SDS, 50 mM Tris-HCl pH 7.5, 5 mM EDTA) in the presence of an equal volume of acid-washed glass beads (Merck Life Science, Milan, Italy) by 10 vortex/ice cycles of 30 sec each. Crude extracts were denatured in SDS sample buffer (40% glycerol, 20% β-mercaptoethanol, 9.2% SDS, 0.25 mM Tris-HCl pH 6.8, 0.01% BBF) and heated for 5 min at 95 °C.
Western blot analysis was conducted with anti-phosphoT172-AMPK antibody (Cell Signaling, Danvers, MA, USA), anti-His antibody (Merck Life Science, Milan, Italy), and anti-TAP antibody (Thermo Fisher Scientific, Waltham, MA, USA). Densitometric analyses were conducted with ImageJ software v1.51 (NIH, http://imagej.nih.gov/ij/, accession date 23 April 2018).
Milanesi R., Tripodi F., Vertemara J., Tisi R, & Coccetti P. (2021). AMPK Phosphorylation Is Controlled by Glucose Transport Rate in a PKA-Independent Manner. International Journal of Molecular Sciences, 22(17), 9483.
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3

ChIP-qPCR Analysis of Protein Binding

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Cross-linking, extract preparation, and sonication were performed as in Ausubel et al. (2010) . Subsequent procedures with anti-TAP antibody (Thermo Scientific) and protein A-coated Dynabeads (Life Technologies) were performed as in van Attikum et al. (2004) (link). qPCR was performed with a Rotor-Gene Q (Qiagen) using the primers listed in Supplemental Table S3. Reported values correspond to the signal for each site relative to an internal control (GIT1) divided by the same ratio of sites within input. The mean and standard deviation of three or more biological replicates are presented. Statistical significance was determined by pairwise Student’s t-tests.
Chen M, & Gartenberg M.R. (2014). Coordination of tRNA transcription with export at nuclear pore complexes in budding yeast. Genes & Development, 28(9), 959-970.
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4

Chromatin Immunoprecipitation of TAP-Tagged Proteins

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Chromatin immunoprecipitation of TAP-tagged Fob1 and Sir2 [80 (link)] and quantitative PCR were performed as described [27 (link)] using Anti-TAP antibody (2 μl per IP, Thermo Scientific) for the immunoprecipitation. For the Sld3-13xMyc and Mcm4-13xMyc ChIP experiments, cells were arrested in G1 with alpha factor prior to the chromatin preparation. Antibody used: anti-Myc, 9E10 from culture supernatant.
Shyian M., Mattarocci S., Albert B., Hafner L., Lezaja A., Costanzo M., Boone C, & Shore D. (2016). Budding Yeast Rif1 Controls Genome Integrity by Inhibiting rDNA Replication. PLoS Genetics, 12(11), e1006414.
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5

Western Blotting of EsxA and TAP-tagged RNases

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Strains for western blotting were lysed as described in the CLASH protocol. Forty mg of protein was resolved on 8% or 15% polyacrylamide gels and transferred to a nitrocellulose membrane. Membranes were blocked for 1 h in blocking solution (5% non-fat milk, 0.1% Tween-20 in PBS). Primary antibody probing was performed overnight at 4 °C using anti-EsxA109 (link) (1/500) or the anti-TAP antibody (1/5000, ThermoFisher) to detect tagged RNases. The membrane was then washed three times in PBST (PBS with 0.1% Tween-20) and visualised using an HRP-linked goat anti-rabbit antibody (1/500, Abcam) and Pearce enhanced chemiluminescence solutions (ThermoFisher).
McKellar S.W., Ivanova I., Arede P., Zapf R.L., Mercier N., Chu L.C., Mediati D.G., Pickering A.C., Briaud P., Foster R.G., Kudla G., Fitzgerald J.R., Caldelari I., Carroll R.K., Tree J.J, & Granneman S. (2022). RNase III CLASH in MRSA uncovers sRNA regulatory networks coupling metabolism to toxin expression. Nature Communications, 13, 3560.
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6

Protein Extraction and Western Blot Analysis

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Samples of cells were lysed using ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% NP-40, 10% glycerol) plus 1 mM PMSF (phenylmethanesulfonylfluoride), proteases inhibitor mix (Complete EDTA free Protease Inhibitor Cocktails Tablets, Roche) and phosphatase inhibitor mix (Cocktail 2, Sigma-Aldrich). Protein concentration was determined using the Bio-Rad protein assay. Western blot analysis was performed using anti-HA antibody (Roche), anti-phosphoT172-AMPK antibody (Cell Signalling), anti-His antibody (Sigma-Aldrich) or anti-TAP antibody (Thermo Fisher Scientific). Densitometric analysis was performed by using the ImageJ software (NIH, http://imagej.nih.gov/ij/).
Tripodi F., Fraschini R., Zocchi M., Reghellin V, & Coccetti P. (2018). Snf1/AMPK is involved in the mitotic spindle alignment in Saccharomyces cerevisiae. Scientific Reports, 8, 5853.
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7

Quantifying SUMOylated Ntg1 Variants

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The steady-state level of each Ntg1-TAP fusion protein variant was assessed by immunoblotting whole cell lysates with the rabbit polyclonal anti-TAP antibody (1:3,333 dilution, Open Biosystems) to determine the relative level of differentially modified Ntg1 products. An anti-3-phosphoglycerate (PGK) antibody (1:10,000 dilution; Invitrogen) was used as a control determine the relative level of protein lysate loaded into each lane.
The analysis of immunoblots was performed utilizing the ECL Plex immunoblotting detection system (Amersham), the Typhoon Trio variable mode imager (GE Healthcare), and the ImageQuant TL software package (GE Healthcare). To quantify the percentage of modified Ntg1-TAP, the ratio of modified Ntg1 bands to total Ntg1 signal (including modified and unmodified) was determined for wildtype Ntg1 and each lysine to arginine amino acid substitution variant of Ntg1. Previous work demonstrates that modified Ntg1 contains at least one covalently linked SUMO and the size of higher bands is consistent with multiple SUMO additions(24 (link)). Standard error of the mean was calculated for each. The two-sample Student’s t-test was employed to test for significance (α=0.05).
Swartzlander D.B., McPherson A.J., Powers H.R., Limpose K.L., Kuiper E.G., Degtyareva N.P., Corbett A.H, & Doetsch P.W. (2016). Identification of SUMO Modification Sites in the Base Excision Repair Protein, Ntg1. DNA repair, 48, 51-62.
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8

Tandem Affinity Purification of FACT Complex

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FACT was purified from an Spt16-TAP (Tandem Affinity Purification) yeast strain using the TAP purification procedure (32 (link)). For each purification, 2 L of yeast culture (OD600 of ∼3.0) was spun down and cells were lysed in NP-40 buffer (15 mM Na2HPO4, 10 mM NaH2PO4, 1% NP-40, 150 mM NaCl, 2 mM EDTA, 1 mM DTT, 1× Protease inhibitor cocktail) by bead beating. After centrifugation, the cleared cell lysate was incubated with IgG Sepharose fast flow beads (GE Healthcare). FACT bound to the beads was released by digestion with Tobacco Etch Virus protease (TEV protease, Invitrogen) overnight at 4°C. FACT was subsequently bound by Calmodulin Sepharose beads (GE Healthcare) in the presence of 3 mM CaCl2. After extensive washes, FACT was eluted with the elution buffer containing 20 mM EGTA. The presence of FACT in each eluted fraction was checked by western blotting with an anti-TAP antibody (Open Biosystems). The peak fractions were combined and FACT was concentrated using Amicon Ultra-0.5 ml concentration columns (Millipore). EGTA was diluted during the concentration process.
Mao P., Kyriss M.N., Hodges A.J., Duan M., Morris R.T., Lavine M.D., Topping T.B., Gloss L.M, & Wyrick J.J. (2016). A basic domain in the histone H2B N-terminal tail is important for nucleosome assembly by FACT. Nucleic Acids Research, 44(19), 9142-9152.
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