Anti shp2

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Anti-SHP2 is a lab equipment product that functions as an antibody specific to the SHP2 (Src Homology 2 domain-containing Phosphatase 2) protein. SHP2 is a protein tyrosine phosphatase that plays a crucial role in various cellular signaling pathways. The Anti-SHP2 product can be used for the detection and study of SHP2 in research applications.

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20 protocols using anti shp2

Immunofluorescence Staining of Germ Cell Markers

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The prepared sections were blocked with 3% (w/v) bovine serum albumin (BSA; ZSbio) in PBST (0.1% [v/v] Triton X-100 in PBS) for 1 h at room temperature and then incubated with the following primary antibodies overnight at 4°C: anti-Sycp3 (1:200, Abcam, Cambridge, MA, USA), anti-synaptonemal complex protein 1 (Sycp1; 1:200, Abcam), anti-Vasa (1:500, Abcam), anti-c-kit (1:500, Abcam), anti-Shp2 (1:200, Santa Cruz Biotechnology), anti-Plzf (1:500, Santa Cruz Biotechnology), anti-cleaved caspase 3 (1:200, Cell Signaling Technology, Boston, MA, USA), anti-Dmc1 (1:200, Abcam), anti-Smc3 (1:500, Abcam), and anti-DNA repair recombinase rad51 (Rad51; 1:200, Invitrogen). After being washed three times with PBST, the samples were incubated with the following secondary antibodies at a 1:200 dilution for 1 h at 37°C: Alexa Fluor 594/488-labeled anti-rabbit or anti-mouse IgG (YEASEN, Shanghai, China). The slides were subsequently washed three times in PBST and mounted with Vectashield containing 4’-6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA).

Li Y., Liu W.S., Yi J., Kong S.B., Ding J.C., Zhao Y.N., Tian Y.P., Feng G.S., Li C.J., Liu W., Wang H.B, & Lu Z.X. (2019). The role of tyrosine phosphatase Shp2 in spermatogonial differentiation and spermatocyte meiosis. Asian Journal of Andrology, 22(1), 79-87.

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Immunofluorescence Staining of Fixed Cells

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Fixed cell samples were exposed to a second round of fixation in PHEM buffer with 2% PFA and 3% BSA (bovine serum albumin) for 10 min at room temperature in order to reduce non-specific adsorption of probing antibodies to the coverslip. Samples were washed 3 times with PHEM buffer, permeabilised with 0.1% saponin in PHEM buffer for 15 min, then washed 3 more times with PHEM buffer. Samples were then quenched with 100mM glycine for 20 min and blocked with 6% BSA in PHEM buffer for 1 h before washing 3 times with PHEM buffer. Samples were then incubated with the appropriate primary rabbit antibody (anti-SHP1, Santa Cruz Biotechnology, sc-287; anti-SHP2, Santa Cruz Biotechnology, sc-280; anti-pY493 ZAP70, Cell Signaling Technology, 2704; anti-pT538 PKCθ, Cell Signaling Technology, 9377) in PHEM buffer with 0.02% saponin, 3% BSA for 1 h. Washing was performed 3 times with PHEM buffer, 0.1% saponin, 3% BSA with 2 min between each wash, before incubating with goat anti-rabbit F(ab’)2 conjugated to AlexaFluor 568 (ThermoFisher Scientific, A-21069) in PHEM buffer with 0.02% saponin, 3% BSA for 45 min. Samples were finally washed 5 times with PHEM buffer, 0.1% saponin, 3% BSA with 2 min between each wash before imaging. A sample stained using only secondary antibody was included in each experiment as a background control.

Mintz M.A., Felce J.H., Chou M.Y., Mayya V., Xu Y., Shui J.W., An J., Li Z., Marson A., Okada T., Ware C.F., Kronenberg M., Dustin M.L, & Cyster J.G. (2019). The HVEM-BTLA Axis Restrains T Cell Help to Germinal Center B Cells and Functions as a Cell-Extrinsic Suppressor in Lymphomagenesis. Immunity, 51(2), 310-323.e7.

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Investigating Anti-Abl Signaling Pathway

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SBF‐1 is a synthetic steroidal glycoside, as described previously.¹⁵, ¹⁶ Anti‐c‐Abl, anti‐p‐Bcr, anti‐p‐STAT5, anti‐STAT5, anti‐p‐SHP‐2, anti‐c‐Cbl, anti‐HA‐tag, anti‐myc‐tag, and anti‐Beclin 1 antibodies were purchased from Cell Signaling Technology. Anti‐SHP‐2, anti‐GAPDH, anti‐PTP1B, and anti‐ ubiquitin antibodies were from Santa Cruz Biotechnology. Anti‐p62, anti‐ATG5, and anti‐phosphotyrosine antibodies were from Abcam. MG132 and bafilomycin A1 (Baf A1) were from Selleck Chemicals. Anti‐Flag‐tag antibody, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), imatinib, 3‐Methyladenine (3‐MA), chloroquine (CQ), 4′,6‐diamidino‐2‐phenylindole (DAPI), and Oridonin were from Sigma‐Aldrich. The lysosome‐specific dye LysoTraker Red, Lipofectamine™ LTX Reagent, Lipofectamine 2000, and Lipofectamine RNAi MAX were from Life Technologies. SuperSep Phos‐tag™ was from Wako. The RediPlate 96 EnzChek Tyrosine Phosphatase Assay Kit was from Thermo Fisher Scientific.

Elgehama A., Wang Y., Yu Y., Zhou L., Chen Z., Wang L., Sun L., Gao J., Yu B., Shen Y, & Xu Q. (2022). Targeting the PTP1B‐Bcr‐Abl1 interaction for the degradation of T315I mutant Bcr‐Abl1 in chronic myeloid leukemia. Cancer Science, 114(1), 247-258.

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Antibody usage for immunodetection

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Anti-hemagglutinin (HA) antibody (3F10) (Roche, Basel, Switzerland) was used as the primary antibody for immunoprecipitation, immunoblotting and immunostaining. Anti-FLAG (M2) (SIGMA, St. Louis, MO, USA) and anti-Omni (Santa Cruz, Dallas, TX, USA) antibodies were used for immunoprecipitation and immunoblotting. Anti-SHP2 (Santa Cruz), anti-Myc (9E10) (Santa Cruz), anti-phosphotyrosine (4G10) (Millipore, Billerica, MA, USA) and anti-Actin (Santa Cruz) antibodies were used for immunoblotting. Anti-ZO-1 antibody (Invitrogen, Carlsbad, CA, USA) was used for immunostaining.

Hashi K., Murata-Kamiya N., Varon C., Mégraud F., Dominguez-Bello M.G, & Hatakeyama M. (2014). Natural variant of the Helicobacter pylori CagA oncoprotein that lost the ability to interact with PAR1. Cancer Science, 105(3), 245-251.

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Dissecting CD112R Tyrosine Phosphorylation

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Mutants for the two tyrosines (Y233 and Y293) in human CD112R intracellular domain were made by changing respective tyrosine to phenylalanine. Assays for pervanadate-induced tyrosine phosphorylation were performed as previously described (Zhu et al., 2013 (link)). In brief, HEK293T cells transfected with individual plasmid were incubated with pervanadate for 10 min before lysis. Cell lysates were immunoprecipitated with CD112R mAb (clone 2H6) and protein G magnetic beads (Invitrogen). After SDS-PAGE, blots were analyzed for phosphotyrosine using 4G10 (EMD Millipore) or CD112R mAb (clone 2H6).
Molt4 cell, a T cell leukemia cell line expressing CD112R, was used to analyze the association of CD112R with possible phosphatases. In brief, Molt4 cells were incubated with pervanadate before being lysed in radioimmunoprecipitation assay buffer. Cell lysate was immunoprecipitated with anti-CD112R (clone 2H6). The possible associated phosphatases were detected by the following antibodies: anti–SHP-1 (Santa Cruz Biotechnology, Inc.), anti–SHP-2 (Santa Cruz Biotechnology, Inc.), and anti-SHIP (Santa Cruz Biotechnology, Inc.).

Zhu Y., Paniccia A., Schulick A.C., Chen W., Koenig M.R., Byers J.T., Yao S., Bevers S, & Edil B.H. (2016). Identification of CD112R as a novel checkpoint for human T cells. The Journal of Experimental Medicine, 213(2), 167-176.

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Western Blot Analysis of Signaling Proteins

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The indicated cells were lysed in lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.5], 1% NP-40, 1 mM EDTA, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM Na₃VO₄, 1 mM NaF, 1 mM PMSF, and 0.1 mg/mL leupeptin/aprotinin) on ice for 30 min, centrifugated at 15,000×g for 30 min, and the supernatants were collected for Western blotting. The primary antibodies were incubated with the membrane overnight at 4 °C. Horseradish peroxidase-conjugated goat anti-rabbit-mouse or rabbit IgG secondary antibodies (Beyotime, Haimen, China) were incubated with the nitrocellulose blotting membranes (GE Healthcare Life science, Boston, Massachusetts) for 1 h at room temperature in the next morning. Images were obtained using a chemiluminescence (ECL) detection system (ProteinSimple, San Jose, CA). Quantified band intensities were normalized using β-actin as housekeeping protein. Blots were scanned with a Tanon imaging system (5200, Shanghai, China). The primary antibodies used included anti-STAT3, anti-ERK1/2, anti-p-STAT3, anti-p-ERK1/2, anti- PI3Kγ, anti-HRAS, anti-SAP18, anti-JAK, anti-p-JAK, anti-SHP2, anti-p-SHP2, anti-PARP1, anti-PAR, and anti-β-actin, which were all purchased from Santa Cruz Biotechnology and Cell Signaling Technology.

Han X., Shi H., Sun Y., Shang C., Luan T., Wang D., Ba X, & Zeng X. (2019). CXCR2 expression on granulocyte and macrophage progenitors under tumor conditions contributes to mo-MDSC generation via SAP18/ERK/STAT3. Cell Death & Disease, 10(8), 598.

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EGF-Induced ERK1/2 Phosphorylation

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ERK1/2 phosphorylation experiments were carried out on transfected HEK293T cells that had been seeded in 6-well plates (70 percent to 80 percent confluence). Following transfection, cells were incubated for 48 h under serum-starved conditions and treated with 20 ng/mL epidermal growth factor (EGF) for 5 or 15 min or left unstimulated. Using a radioimmunoprecipitation technique, the total protein was recovered from the lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA). Transfer membranes containing equal amounts of proteins were treated overnight at 4°C with specified primary antibodies and then at 37°C for two hours with secondary antibodies. The primary antibodies and dilutions were as follows: anti-SHP2 (1 : 3000, Santa Cruz Biotechnology, Dallas, TX, USA), anti-pERK1/2 (1 : 1000, Cell Signaling Technology, Danvers, MA, USA), and anti-Vinculin (1 : 5000, Abmart, Shanghai, China). Anti-rabbit and anti-mouse horseradish peroxidase- (HRP-) conjugated secondary antibodies were employed at 1 : 5000. Band density was quantified with ImageJ software.

Xu Z.Q., Chen W.C., Li Y.J., Suo M.J., Tian G.X., Sheng W, & Huang G.Y. (2022). PTPN11 Gene Mutations and Its Association with the Risk of Congenital Heart Disease. Disease Markers, 2022, 8290779.

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Immunostaining of Fixed Cell Samples

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Fixed cell samples were exposed to a second round of fixation in PHEM buffer with 2% PFA and 3% BSA (bovine serum albumin) for 10 min at room temperature in order to reduce non-specific adsorption of probing antibodies to the coverslip. Samples were washed 3 times with PHEM buffer, permeabilized with 0.1% saponin in PHEM buffer for 15 min, then washed 3 more times with PHEM buffer. Samples were then quenched with 100mM glycine for 20 min and blocked with 6% BSA in PHEM buffer for 1 h before washing 3 times with PHEM buffer. Samples were then incubated with the appropriate primary rabbit antibody (anti-SHP1, Santa Cruz Biotechnology, sc-287; anti-SHP2, Santa Cruz Biotechnology, sc-280; anti-pY493 ZAP70, Cell Signaling Technology, 2704; anti-pT538 PKCθ, Cell Signaling Technology, 9377) in PHEM buffer with 0.02% saponin, 3% BSA for 1 h. Washing was performed 3 times with PHEM buffer, 0.1% saponin, 3% BSA with 2 min between each wash, before incubating with goat anti-rabbit F(ab’)2 conjugated to AlexaFluor 568 (ThermoFisher Scientific, A-21069) in PHEM buffer with 0.02% saponin, 3% BSA for 45 min. Samples were finally washed 5 times with PHEM buffer, 0.1% saponin, 3% BSA with 2 min between each wash before imaging. A sample stained using only secondary antibody was included in each experiment as a background control.

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Immunoblot Analysis of Signaling Proteins

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Immunoblot assay was performed as described previously^{60 (link)}. Briefly, proteins extracted in lysis buffer were separated by SDS–polyacrylamide gel electrophoresis and electrophoretically transferred onto polyvinylidene difluoride membranes. The membranes were probed with antibodies overnight at 4 °C, and then incubated with a horseradish peroxidase-coupled secondary antibody. Detection was performed using a LumiGLO chemiluminescent substrate system. The following antibodies were used at indicated dilutions: anti-SHP2 (Santa Cruz Biotechnology, sc-7384), anti-SOX9 (Abcam, ab76997), anti-pSmad1/5 (Cell Signaling Technology, 9516), anti-p-Erk1/2 (Cell Signaling Technology, 4370), anti-ACTIN (Abmart, M20010).

Shao F., Liu Q., Zhu Y., Fan Z., Chen W., Liu S., Li X., Guo W., Feng G.S., Yu H., Xu Q, & Sun Y. (2021). Targeting chondrocytes for arresting bony fusion in ankylosing spondylitis. Nature Communications, 12, 6540.

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Comprehensive Immunophenotyping and Signaling Analysis

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For flow cytometry and ImageStream analysis: phycoerythrin (PE)-conjugated LAG3 (eBioscience), PD1-botin (RMP1–14, Biolegend), PD1-BV421 (Biolegend), (perCP-conjugated CD8a (eBioscience), and FITC-conjugated Thy1.1. For immunoprecipitation: rat-anti-mLAG3 (BD, Pharmingen monoclonal), rat-anti-mPD-1 (a gift from T. Honjo, or eBioscience, Rat IgG2), control rat-IgG1 and IgG2 (Biolegend), control rabbit IgG (Santa-Cruz or Biolegend), rabbit anti-SHP2 (Santa-Cruz or AbCam). For Western blot analysis: goat anti-LAG-3 mAb (C9B7W, R&D Systems), goat anti-PD1 mAb (R&D Systems), anti-SHP1 (AbCam), anti-SHP2 (Santa-Cruz). All the secondary antibodies were from KPL. For confocal microscopy: rat-anti-mPD-1 (RMP1–14, from Tasuku Honjo, Osaka University), goat-anti-LAG3 (R&D Systems), rabbit-anti-EEA1 (Cell Signaling), rabbit-anti-Rab11b (Cell Signaling), rabbit-anti-LAMP1 (Cell Signaling), mouse-anti-TGN38 (BD Biosciences), rabbit-anti-tubulin-r (Biolegend), pLck (Cell Signaling). The following secondary antibodies, absorbed against cross-reactive species, were used: goat-Cy3 and mouse-DyLight-649 were from Jackson ImmunoResearch and rat-Alexafluo-488 and rabbit-Alexafluo-647 antibodies were from Molecular Probe.

Huang R.Y., Eppolito C., Lele S., Shrikant P., Matsuzaki J, & Odunsi K. (2015). LAG3 and PD1 co-inhibitory molecules collaborate to limit CD8+ T cell signaling and dampen antitumor immunity in a murine ovarian cancer model. Oncotarget, 6(29), 27359-27377.

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