Ab9337

Manufactured by Abcam

Ab9337 is a monoclonal antibody that recognizes a specific target antigen. The product is intended for use in various research applications, but a detailed description cannot be provided while maintaining an unbiased and factual approach. The core function of this product is to specifically bind to the target antigen, but no further interpretation or extrapolation on its intended use can be made.

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Lab products found in correlation

5 protocols using ab9337

Co-immunoprecipitation of Flag- and Myc-tagged Proteins

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For co-immunoprecipitation assays, HEK293T cells were harvested and lysed with either TNE lysis buffer [10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA and 0.5% Nonidet P-40] or RIPA buffer (R0010, Solarbio) containing a protease inhibitor mixture (1697498001, Roche). Lysates were incubated with anti-Flag-agarose beads (A2220, Sigma-Aldrich) or protein A-Sepharose beads (101041, Invitrogen) at 4°C for 4 h. Beads were washed four times with TNE or RIPA buffer, and bound proteins were then separated by SDS-PAGE and visualized using western blots.
For immunoblotting experiments, we used the following affinity-purified antibodies: anti-Flag (1:1000; Cell Signaling Technology, 2368S), anti-Myc (1:3000; M047-3, MBL), anti-HA (1:3000; CW0092A, CW), anti-β-tubulin (1:5000, CW0098M, CWBIO), anti-p-Thr (1:300; ab9337, Abcam) and anti-p-Ser (1:250; ab9332, Abcam).

Liu J., Zhang M., Dong H., Liu J., Mao A., Ning G., Cao Y., Zhang Y, & Wang Q. (2022). Chemokine signaling synchronizes angioblast proliferation and differentiation during pharyngeal arch artery vasculogenesis. Development (Cambridge, England), 149(23), dev200754.

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Phospho-STAT3 T622 Antibody Production

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Rabbit polyclonal antibody recognizing phosphorylated STAT3 T622 was customized from Boer Biotechnology. To prepare antibody recognizing STAT3 pT622, rabbits were treated with peptide containing STAT3 pT622. Nonmodified peptide immobilized on an affinity column was used to remove the antibodies recognizing nonphosphorylated STAT3, and STAT3 pT622 peptide immobilized on an affinity column was used to associate with and isolate the antibodies. The eluted antibodies were then concentrated.
Antibodies recognizing TAZ (#83669), STAT3 (#9139), MST2 (#3952), MST1 (#14946), YAP1 (#12395), SAV1 (#13301), and STAT3‐pY705 (#9145) were obtained from Cell Signaling Technology. Antibody simultaneously recognizing LATS1 and LATS2 (BS‐4081R) was obtained from Bioss. Antibodies recognizing LATS1/2 pT1079/T1041 (ab111344), Ki67 (ab16667), YAP1 (ab205270), HA (ab9110), tubulin (ab7291), Flag (ab205606), GST (ab36415), YAP1 pS127 (ab76252), phospho‐Thr (ab9337), JAK2 (ab108596), and recombinant IL‐6 protein (ab9627) were purchased from Abcam. Anti‐Flag agarose beads were obtained from Sigma. XMU‐MP‐1 was obtained from MedChemExpress. [γ‐³²P]‐ATP was obtained from MP Biomedicals.

Tang Q., Fang J., Lai W., Hu Y., Liu C., Hu X., Song C., Cheng T., Liu R, & Huang X. (2022). Hippo pathway monomerizes STAT3 to regulate prostate cancer growth. Cancer Science, 113(8), 2753-2762.

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Phosphorylation of Recombinant PKM2 by AKT1

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A phosphorylation assay was performed in 1X kinase buffer (Cell Signaling Technology, 9802) containing 3.3 μM ATP (Cell Signaling Technology, 9804) with 1 μg of wild-type or S202A mutant GST-PKM2 and 200 ng of active AKT1 (Millipore, 14-276). After incubation for 1 h at 30°C, the reaction was stopped by adding SDS sample buffer and then the samples were subjected to western blot analysis using anti-phospho-serine (Abcam, ab9332) or anti-phospho-threonine (Abcam, ab9337) antibodies. For the autoradiogram analysis, 10 μCi of [γ-32p]ATP (Perkin Elmer, NEG002A250UC) was added to the reaction. The analyzed SDS-PAGE gel was dried and exposed to X-ray film (Kodak) for 3 days.

Park Y.S., Kim D.J., Koo H., Jang S.H., You Y.M., Cho J.H., Yang S.J., Yu E.S., Jung Y., Lee D.C., Kim J.A., Park Z.Y., Park K.C, & Yeom Y.I. (2016). AKT-induced PKM2 phosphorylation signals for IGF-1-stimulated cancer cell growth. Oncotarget, 7(30), 48155-48167.

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Comprehensive Protein Analysis Workflow

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Total cell lysates were obtained using RIPA buffer (P0013; Beyotime). WB was conducted as previously described.²⁸ The primary antibodies used are as follows: phosphothreonine (p‐Thr, ab9337; Abcam), phosphoserine (p‐Ser, ab9332; Abcam), eIF4GI (2858s; Cell Signaling Technology), MDA5 (5321s; Cell Signaling Technology), GFP (2955s; Cell Signaling Technology), VP1 (PAB7631‐D01P; Abnova), VP1(GTX633390; CeneTex), His (Biodragon, B1004), total ERK1/2 (t‐ERK1/2, 9102s; Cell Signaling Technology), phospho‐ERK1/2 (p‐ERK1/2, 9106s), and β‐actin (BS6007 M; BioWorld). Specific bands were visualized using enhanced chemiluminescence. The grayscale intensity of each WB band was quantified using ImageJ (version 1.52a).

Wang Y., Zou W., Niu Y., Wang S., Chen B., Xiong R., Zhang P., Luo Z., Wu Y., Fan C., Zhong Z., Xu P, & Peng Y. (2022). Phosphorylation of enteroviral 2Apro at Ser/Thr125 benefits its proteolytic activity and viral pathogenesis. Journal of Medical Virology, 95(1), e28400.

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Phosphorylation of CATs by PC1

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To obtain phosphorylated CATs, WT (Kitaake) seedlings at the 3-leaf stage were treated with 140 mM NaCl for 30 min, and the phosphorylated CATs proteins were immunoprecipitated with an anti-CAT antibody. Then, recombinant GST-PC1 or GST purified from E. coli with glutathione agarose beads (Roche, CH) was added to an in vitro phosphate reaction buffer [50 mM HEPES, pH 7.4, 50 mM NaCl, 0.1% (v/v) Triton X-100, and 1 mM DTT] with a different metal ion (5 mM EDTA, 10 mM MgCl 2 , or 10 mM MnCl 2 ) for 90 min at 30 °C to verify the ion requirements for PC1. The phosphorylation state of CATs was determined by immunoblotting using antiphospho-serine (pSer, Abcam, ab9332, dilution 1:1,000), anti-phospho-threonine (pThr, Abcam, ab9337, dilution 1:1,000), or anti-phospho-tyrosine (pTyr, Abcam, ab17302, dilution 1:1,000) antibodies.

Liu C., Lin J.Z., Wang Y., Tian Y., Zheng H.P., Zhou Z.K., Zhou Y.B., Tang X.D., Zhao X.H., Wu T., Xu S.L., Tang D.Y., Zuo Z.C., He H., Bai L.Y., Yang Y.Z, & Liu X.M. (2023). The protein phosphatase PC1 dephosphorylates and deactivates CatC to negatively regulate H2O2 homeostasis and salt tolerance in rice. The Plant cell, 35(9).

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