Mouse anti gst

Manufactured by Merck Group

Sourced in United States, United Kingdom

Mouse anti-GST is a laboratory reagent used to detect and capture glutathione S-transferase (GST) fusion proteins. It is a monoclonal antibody produced in mice that specifically binds to the GST tag, which is commonly used to purify recombinant proteins expressed in various expression systems.

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Anti-GST (56 citations) Mouse anti-GST antibody (2 citations)

8 protocols using mouse anti gst

Antibody Reagents for Western Blot

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The mouse anti-FLAG (Sigma-Aldrich, USA), mouse anti-Myc (Sigma-Aldrich, USA), rabbit anti-ATP6V1A (Sigma-Aldrich, USA), mouse anti-β-actin (Proteintech, China), mouse anti-HA epitope tag (Proteintech, China), mouse anti-GST (Sigma-Aldrich, USA), mouse anti-His epitope tag (Proteintech, China), rabbit IgG (Solarbio, China) and the secondary antibodies (HRP-conjugated anti-mouse IgG antibody [Sigma-Aldrich, USA], HRP-conjugated anti-rabbit IgG antibody [Genescript, China], FITC-conjugated goat anti-mouse IgG antibody [ZSGB. Bio, China], Alexa Fluor 647-labeled goat anti-rabbit IgG (H + L) [Beyotime, China]) and gold-labeled goat anti-mouse IgG antibody (Sigma-Aldrich, USA) were purchased from the indicated vendors. Murine sera against M, P, or G protein were produced in our laboratory.

Liu X., Li F., Zhang J., Wang L., Wang J., Wen Z., Wang Z., Shuai L., Wang X., Ge J., Zhao D, & Bu Z. (2020). The ATPase ATP6V1A facilitates rabies virus replication by promoting virion uncoating and interacting with the viral matrix protein. The Journal of Biological Chemistry, 296, 100096.

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Antibody Validation and Characterization

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The following antibodies were used: mouse anti-GST (Sigma), rat anti-mCherry (Invitrogen), rabbit anti-Axin1 (C76H11) (Cell Signaling Technologies; CST), rabbit anti-Lrp6 (C47E12) (CST), rabbit anti-phospho-Lrp6 (Ser1490) (CST), mouse anti-Myc (9E10) (DSHB), rabbit anti-Myc (71D10) (CST), mouse anti-β-tubulin (Sigma), mouse anti-GAPDH (Abcam), rat anti-HA (Roche), rabbit anti-GFP (Life Technologies), mouse anti-GFP (Roche), chicken anti-GFP (Life Technologies), rabbit anti-FLAG tag (CST).

Mah K.M., Houston D.W, & Weiner J.A. (2016). The γ-Protocadherin-C3 isoform inhibits canonical Wnt signalling by binding to and stabilizing Axin1 at the membrane. Scientific Reports, 6, 31665.

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Molecular Dynamics of Ebolavirus VP40 Protein

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HEK293T were maintained in Dulbecco’s (Radnor, PA, USA) modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and penicillin (100 U/mL)–streptomycin (100 μg/mL) at 37 °C in a humidified 5% CO₂ incubator. Plasmids expressing wild-type eVP40 (eVP40-WT) and eVP40-ΔPT/PY (PTAPPEY deletion mutant) were described previously [20 (link),22 (link)]. Plasmids expressing FLAG-tagged mVP40-WT and PPxY mutant mVP40 (P > A) were kindly provided by S. Becker (Institut fur Virologie, Marburg, Germany). Plasmids expressing SMURF2-FLAG-WT or the enzymatically inactive SMURF2-FLAG-C716A mutant were purchased from Addgene (Watertown, MA, USA, catalog numbers 11746 and 11747). SMURF2-specific or random small interfering RNAs (siRNAs) were purchased from Dharmacon (Lafayette, CO, USA, catalog number L-007194-00-0005 and D-001810-10-20). Mouse anti-FLAG monoclonal antibody was purchased from Fitzgerald Industries International (Acton, MA, USA, catalog number 10R-2139), rabbit anti-SMURF2 was purchased from Cell Signaling Technology (Danvers, MA, USA, catalog number 12024S), and mouse anti-GST (catalog number SAB4200237) as well as mouse anti-β-actin (catalog number A1978) antisera were obtained from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-eVP40 antibody was purchased from IBT Bioservices (Rockville, MD, USA, catalog number 0301-010).

Shepley-McTaggart A., Schwoerer M.P., Sagum C.A., Bedford M.T., Jaladanki C.K., Fan H., Cassel J, & Harty R.N. (2021). Ubiquitin Ligase SMURF2 Interacts with Filovirus VP40 and Promotes Egress of VP40 VLPs. Viruses, 13(2), 288.

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Antibody Immunofluorescence and Western Blotting

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The following antibodies
were used for either
immunofluorescence staining or Western blotting purposes: goat anti-NEK7
(sc50756), goat (sc50763) or mouse (sc1004) anti-NEK9, goat anti-α-tubulin
(TUBA) (sc8035) (all from Santa Cruz Biotechnology); mouse anti-NEK7
(ab68060), rabbit anti-NEK7 (ab96538), goat anti-PLEKHA8 (ab38748),
rabbit anti-β-tubulin (TUBB) (ab15568), rabbit anti-RGS2 (ab36561),
rabbit anti-MNAT1 (ab65125), rabbit anti-ANKS1B (ab116083), mouse
anti-CC2D1A (ab68302), and mouse anti-pericentrin (PCNT) (ab28144)
(all from Abcam). Mouse anti-His₅ (34660, QIAGEN), mouse
anti-GST (made in house), mouse anti-FLAG (F1804, Sigma-Aldrich),
rabbit anti-phosphothreonine (71-8200, Invitrogen), mouse anti-PLK1
(ab17057), goat anti-SMC1 (sc-21078), and goat anti-SMC3 (sc-8198)
antibodies were used for Western blotting. Secondary antibodies for
immunofluorescence staining were obtained from the following sources:
chicken anti-goat, anti-mouse, or anti-rabbit Alexa Fluor 488 or donkey
anti-goat, anti-mouse, or anti-rabbit Alexa Fluor 546 (all from Life
Technologies, Inc.); horseradish peroxidase (HRP)-conjugated anti-mouse
was obtained from (Calbiochem) and HRP-linked anti-goat or anti-rabbit
secondary antibodies for Western blotting (WB) purposes were obtained
from Sigma-Aldrich.

de Souza E.E., Meirelles G.V., Godoy B.B., Perez A.M., Smetana J.H., Doxsey S.J., McComb M.E., Costello C.E., Whelan S.A, & Kobarg J. (2014). Characterization of the Human NEK7 Interactome Suggests Catalytic and Regulatory Properties Distinct from Those of NEK6. Journal of Proteome Research, 13(9), 4074-4090.

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Immunoprecipitation and Immunoblotting of rEag1

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Immunoprecipitation and immunoblotting were performed as described previously 6, 20. In brief, transfected cells were solubilized in ice‐cold IP buffer [20 mm Tris–HCl, pH 7.4, 150 mm NaCl, 10 mm Na₂HPO₄, 1% Triton X‐100, 0.5% Na‐deoxycholate, 0.1% SDS, 1 mm EDTA, and 1 mm phenylmethylsulfonyl fluoride (PMSF)]. Where indicated, 2 mm EGTA or 2 mm CaCl₂ was added in lieu of EDTA. Solubilized lysates were incubated for 16 h at 4 °C with protein A and G sepharose beads (GE Healthcare Biosciences, Marlborough, MA, USA) precoated with the indicated rabbit and mouse antibodies, respectively. Protein samples were separated on 7.5–15% SDS/PAGE, transferred to nitrocellulose membranes, followed by immunoblotting. For detecting centrin signal, membranes were fixed with 0.2% glutaraldehyde prior to primary antibody incubation. Input represents 5% of the total protein used for immunoprecipitation. The antibodies include mouse anti‐β‐actin (Sigma, St. Louis, MO, USA), mouse anti‐centrin (Millipore, Billerica, MA , USA), rabbit anti‐rEag1 (Alomone, Jerusalem, Israel), rabbit anti‐GFP (Abcam, Eugene, OR, USA), mouse anti‐GST (Sigma), mouse IgG (Sigma), mouse anti‐Myc (clone 9E10), mouse anti‐PSD‐95 (NeuroMab, Davis, CA, USA), and mouse anti‐synaptophysin. Results shown are representative of at least three independent experiments.

Hsu P., Chiu Y., Lin T, & Jeng C. (2016). Ca2+‐binding protein centrin 4 is a novel binding partner of rat Eag1 K+ channels. FEBS Open Bio, 6(4), 349-357.

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Detailed Western Blotting and Immunofluorescence Protocols

Cited 2 times

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For Western blotting the following antibodies were used at the concentrations indicated: mouse anti-Rab27a, 1∶10000 [22] (link); rabbit anti R3G, 1∶5000 (a kind gift of J. Myoshi and Y. Takai); mouse anti-GST, 1∶10000 (Sigma G1160); mouse anti-tubulin, 1∶5000 (Sigma T6557); mouse anti-ATP1a1, 1∶5000 (Abcam ab7671); rabbit anti-His, 1∶1000 (Abcam ab9108); mouse anti Pmel17, 1∶500 (Dako M0634); goat anti-GPNMB, 1∶1000 (R&D Systems AF2330); rabbit anti-Annexin A2, 1∶5000 (a kind gift from J. Ayala-SanMartin CNRS/UPMN/ENS).
For immunofluorescence the following antibodies were used at the concentrations indicated: rabbit anti-Rab27a, 1∶200 (purified by R. Singh, Imperial College, UK); mouse anti-ATP1a1, 1∶200 (Thermo MA1-16731); rabbit anti-Rab5a, 1∶50 (Abcam ab18211); rabbit anti-Rab38, 1∶100 (purified by C. Wasmeier, Imperial College, UK [33] (link)); rabbit anti-Mlph (purified by A. Hume, Imperial College, UK [34] (link))
For immunoprecipitation the monoclonal anti-HA (Roche 12CA5) and anti-FLAG (Sigma F3165) were used.

Booth A.E., Tarafder A.K., Hume A.N., Recchi C, & Seabra M.C. (2014). A Role for Na+,K+-ATPase α1 in Regulating Rab27a Localisation on Melanosomes. PLoS ONE, 9(7), e102851.

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BES1-HSFA1A Interaction Analysis

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For in vitro pull‐down experiments, recombinant BES1‐GST or GST alone (as a control) was incubated with GST beads for 1 h in the reaction buffer (150 mM NaCl and 50 mM Tris–HCl, pH 8.0). After BES1‐GST or GST was immunoprecipitated and washed, recombinant HSFA1a‐MBP was added and incubated for 1 h more (input sample). Then, five washing steps were performed before the proteins were eluted with 4x SDS buffer (pull‐down sample). Input and pull‐down samples were separated by PAGE and transferred to membranes. The presence of BES1‐GST or GST was verified with a mouse anti‐GST (Sigma‐Aldrich) and co‐immunoprecipitated HSFA1A‐MBP was detected with a mouse anti‐MBP (New England Biolabs) and alkaline phosphatase‐conjugated goat anti‐mouse IgG (Sigma‐Aldrich) as the secondary antibody.
For BiFC assays (Walter et al, 2004 (link)), BES1 was fused to the N‐terminal and HSFA1a to the C‐terminal part of YFP as described previously (Khan et al, 2013 (link)). The constructs were transiently co‐introduced into Nicotiana benthamiana leaves via Agrobacterium‐mediated transformation, and after 2 days of incubation, fluorescence was visualized using confocal microscopy.

Albertos P., Dündar G., Schenk P., Carrera S., Cavelius P., Sieberer T, & Poppenberger B. (2022). Transcription factor BES1 interacts with HSFA1 to promote heat stress resistance of plants. The EMBO Journal, 41(3), e108664.

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Antibody-based Protein Detection Protocols

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Antibodies: Phycolink^® anti-GST R-phycoerythrin-conjugated antibodies (Europa Bioproducts Ltd., Cambridge, UK), rabbit anti-Cldn1 and anti-Cldn4, (Invitrogen, Carlsbad, USA), rabbit polyclonal anti-Cldn6 (Immuno-Biological Laboratories, Fujioka, JP), mouse anti-GST (Sigma-Aldrich, Steinheim, DE), mouse anti-NS5a (ABCAM, Cambridge, UK).
Chemicals: Lucifer Yellow: Sigma-Aldrich Chemie GmbH (München, DE).

Beier L.S., Rossa J., Woodhouse S., Bergmann S., Kramer H.B., Protze J., Eichner M., Piontek A., Vidal-y-Sy S., Brandner J.M., Krause G., Zitzmann N, & Piontek J. (2019). Use of Modified Clostridium perfringens Enterotoxin Fragments for Claudin Targeting in Liver and Skin Cells. International Journal of Molecular Sciences, 20(19), 4774.

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