Cells were lysed in RIPA buffer containing complete mini protease inhibitor (Roche) for 15 min on ice with occasional vortexing. Samples were centrifuged, and cell lysates were mixed with SDS loading buffer and boiled at 95°C for 10 min. Sample were then loaded and separated on 10% SDS-PAGE gels, before transferring onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% milk diluted in PBS containing 0.05% Tween-20 (PBST) for 1 hour, before incubation with primary antibodies at 1:500-1000 dilution overnight at 4°C. Thereafter, membranes were washed and probed with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h before detection with Western Lightning Chemiluminescence (Perkin Elmer). Antibodies used included: anti-EBNA1 (sc-57719, Santa Cruz), anti-EBNA2 (ab90543, Abcam), anti-LMP1 (CS1-4, Dako), anti-LMP2A (MCA2467, Bio-rad), anti-GAPDH (mab374, Merck), anti-mouse IgG-HRP (ThermoFisher Scientific) and anti-rat IgG HRP (Santa Cruz).
Anti rat igg hrp
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-rat IgG HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and quantify rat immunoglobulin G (IgG) in various immunoassays and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Image reader las 3000 mini, by Fujifilm (2 mentions)
Anti rabbit igg hrp, by Santa Cruz Biotechnology (2 mentions)
Anti mouse igg hrp, by Santa Cruz Biotechnology (2 mentions)
Ab90543, by Abcam (1 mentions)
Cs1 4, by Agilent Technologies (1 mentions)
Anti gapdh mab374, by Merck Group (1 mentions)
Anti mouse igg hrp, by Thermo Fisher Scientific (1 mentions)
Complete mini protease inhibitor, by Roche (1 mentions)
Enhanced chemiluminescence system, by GE Healthcare (1 mentions)
β actin clone ac 15, by Merck Group (1 mentions)
Phospho p44 42 mapk, by Cell Signaling Technology (1 mentions)
α β tubulin, by Cell Signaling Technology (1 mentions)
Phospho akt s473, by Abcam (1 mentions)
Lmnb1, by Merck Group (1 mentions)
Phospho mtor, by Cell Signaling Technology (1 mentions)
Ecl system, by GE Healthcare (1 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (1 mentions)
Tissue tek, by Thermo Fisher Scientific (1 mentions)
Hematoxylin eosin, by Merck Group (1 mentions)
Pe conjugated anti cd11b, by BD (1 mentions)
7 protocols using anti rat igg hrp
1
Western Blot Analysis of EBV Proteins
2
Cell Lysis and Immunoblot Analysis
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Cell lysis, SDS‐PAGE and immunoblot analysis were performed as described previously.8 (link) Membranes were incubated with antibodies against the following proteins: BIG3 (diluted 1:250),6 (link) PHB2 (Abcam #ab71970, diluted 1:1000), β‐actin (clone AC‐15, Sigma‐Aldrich #A1978, diluted 1:5000), PARP (CST #9542, diluted 1:500), and apoptosis‐inducing factor (AIF) (clone E‐1, Santa Cruz Biotechnology #sc‐13116, diluted 1:200). After incubation with HRP‐conjugated secondary antibodies (anti‐mouse IgG‐HRP, diluted 1:5000; anti‐rat IgG‐HRP, diluted 1:5000; anti‐rabbit IgG‐HRP, diluted 1:1000 [Santa Cruz Biotechnology]) for 1 hour, the blots were developed with an enhanced chemiluminescence system (GE Healthcare) and scanned using an Image Reader LAS‐3000 Mini (Fujifilm).
3
E-Selectin-Ig Chimera Binding Assay
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Recombinant mouse E‐selectin/CD62E human immunoglobulin Fc chimera (E‐selectin‐Ig chimera, ‘E‐Ig’) was purchased from R&D Systems (Minneapolis, MN, USA). Anti‐CLA monoclonal antibody (mAb), clone HECA‐452, was from BioLegend (San Diego, CA, USA). Anti‐human CD29 mAb was from ImmunoTools (Friesoythe, Germany). Rat anti‐mouse CD62E, anti‐mouse immunoglobulins (Ig) mAb conjugated with horseradish peroxidase (HRP), and anti‐rat Ig conjugated with allophycocyanin (APC) mAbs were from BD Biosciences (San Jose, CA, USA). Anti‐rat IgG‐HRP, anti‐rat IgM‐HRP, and anti‐β‐tubulin mAbs were from Santa Cruz Biotechnology (Dallas, TX, USA). Anti‐human Ig‐fluorescein (FITC) mAb was from Sigma‐Aldrich (St. Louis, MO, USA). CellTrace™ 5,6‐carboxyfluorescein diacetate succinimidyl ester (CFSE) Cell Proliferation Kit was purchased from Molecular Probes (Leiden, Netherlands), Thermo Fisher Scientific (Waltham, MA, USA). Anti‐human cytokeratin, clone AE1/AE3, was from Dako (Santa Clara, CA USA). Anti‐p44/42 MAPK (ERK1/2), phospho‐ERK1/2 (P‐ERK), p38 MAPK, and phospho‐p38 (P‐p38) MAPK mAbs were from Cell Signaling Technology (Danvers, MA, USA).
4
Immunoblot Analysis of Signaling Pathways
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Immunoblot analyses were performed as described previously10 . After SDS-PAGE, the membranes were blocked with 4% BlockAce solution (Dainippon Pharmaceutical) for 3 h and then incubated with antibodies against the following proteins: BIG3 (1:1,000)10 ; PHB2 (1:1,000, Abcam); Akt (1:1,000), phospho-Akt (S473) (587F11, 1:1,000); p44/42 MAPK (1:1,000), phospho-p44/42 MAPK (T202/Y204) (1:1,000); mTOR (1:1,000); phospho-mTOR (S2448) (1:1,000); S6K (1:1,000); phopho-S6K (T389) (1:1,000); α/β-tubulin (1:1,000) (Cell Signalling Technology); and LMNB1 (1:100, Sigma). After incubation with an HRP-conjugated secondary antibody (anti-mouse IgG-HRP, 1:5,000; anti-rat IgG-HRP; 1:5,000; or anti-rabbit IgG-HRP, 1:1,000; Santa Cruz Biotechnology) for 1 h, the blots were developed with an enhanced chemiluminescence (ECL) system (GE Healthcare) and scanned using an Image Reader LAS-3000 mini (Fujifilm). All the experiments were performed in triplicate at a minimum. Full-length images of immunoblots are shown in Supplementary Fig. S5 .
5
Histological Analysis of Murine Lymphoma
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Organs isolated from moribund IgHµ-TLX1TgPrkdcScid/Scid and PrkdcScid/Scid mice as well as healthy IgHµ-TLX1TgPrkdcScid/Scid mice were fixed in 4% PFA overnight, paraffin embedded, sectioned and stained with hematoxylin-eosin (Sigma, St. Louis, MO, USA). A certified pathologist performed all morphological interpretations of tissue sections. Tumors were also cryopreserved in O.C.T. compound (Tissue-Tek; Fisher Scientific, Waltham, MA, USA) for cryosectioning and 5 µm sections were stained with purified rat anti-mouse Thy1.2 (clone 53-2.1; BD Pharmingen). Antibody binding was visualized using an anti-rat IgG-HRP (Santa Cruz Biotechnology) and Peroxidase Substrate Kit DAB (Vector Laboratories, Inc.). Sections were counterstained with hematoxylin 1∶5 (Fluka).
6
Murine Myeloid Cell Immunophenotyping
7
Immunoprecipitation and Glycosylation Analysis of CD44
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PC3 cells were lysed with low salt lysis buffer [20 mM Tris-Cl pH 7.5, 150 mM NaCl, 1% TritonX-100, and complete mini protease inhibitor cocktail (Roche)] and subjected to immunoprecipitation. Five micro gram of F77 antibody were bound to Dynabead protein G magnetic beads (Invitrogen) according to manufacturer’s instructions, then incubated with the lysate for 2 hr. For membrane proteins, we first incubated mAb F77 (5 μg) with PC3 cells, then lysed cells with low salt lysis buffer and precipitated with protein G magnetic beads. The precipitates were then washed three times with the buffer (20 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.01% Tween-20) and boiled for 5 minutes in SDS loading buffer. Samples were analyzed by SDS-PAGE, transferred to Immobilon-P (Millipore) PVDF membranes, and probed using anti-CD44 antibody, and detected with anti-rat IgG HRP (Santa Cruz Biotechnology) and Immobilon Western Chemiluminescent horseradish peroxidase (HRP) Substrate (Millipore). For glycosylation site analysis, 293T cells were transfected with FUT1 and CD44 mutant plasmids using Fugene6. The cells were then lysed with low salt lysis buffer and subjected to SDS PAGE and western blotting as described above. HA-tagged proteins were detected with anti-HA Peroxidase (3F10; Roche) using ECL.
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