The bead clustering experiment was performed using NIH 3T3 cells stably expressing Sra1-YPet and transiently transfected with CD16-CD7-CT-mCherry chimeric receptors (Kolanus et al., 1993 (link)). Receptors were clustered using an anti-CD16 monoclonal mouse antibody (Invitrogen) followed by Dynal beads coated with sheep-anti-mouse IgG (Invitrogen). Fixed cells were imaged using confocal microscopy along Z stacks to find the beads with enriched mCherry signals and blind-scored for enriched Ypet signals.
Sheep anti mouse igg
Manufactured by Thermo Fisher Scientific
Sourced in China, United States
Sheep anti-mouse IgG is a secondary antibody that binds to mouse immunoglobulin G (IgG) proteins. It is used in various immunological techniques, such as Western blotting, ELISA, and immunohistochemistry, to detect and quantify the presence of mouse IgG in samples.
Automatically generated - may contain errors
Lab products found in correlation
Dynabead, by Thermo Fisher Scientific (2 mentions)
Dynal beads, by Thermo Fisher Scientific (1 mentions)
Dynabeads m 280 sheep anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Complete mini protease inhibitor tablet, by Roche (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
35s cysteine, by PerkinElmer (1 mentions)
Anti p62 sqstm1, by Proteintech (1 mentions)
Anti vimentin, by Proteintech (1 mentions)
Anti p akt ser473, by Abcam (1 mentions)
Ecl substrate, by Clinx (1 mentions)
Chemiscope touch, by Clinx (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Anti lc3, by Proteintech (1 mentions)
Genechip scanner gcs3000, by Thermo Fisher Scientific (1 mentions)
Whole genome amplification kit, by Merck Group (1 mentions)
Fluidic station 450, by Thermo Fisher Scientific (1 mentions)
7 protocols using sheep anti mouse igg
1
Receptor Clustering in 3T3 Cells
2
Dynabeads Immunoprecipitation Protocol
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The experiments were performed using 100 μl of Dynabeads M-280 sheep anti-rabbit IgG or sheep anti-mouse IgG (Invitrogen, #112-03D or #112-01D), and the manufacturer’s instructions were followed.
3
Evaluating TFEB and 14-3-3 Interactions
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Cell lysates from corneal fibroblasts were prepared in a radio‐immunoprecipitation assay buffer (150 mmol/L NaCl, 1% NP‐40, 0.5% deoxycholate, 0.1% SDS, 50 mmol/L Tris‐HCl, pH 7.4) containing a protease inhibitor (Complete Mini Protease Inhibitor Tablet, Roche #1836170). Supporting information provides more additional detail methods (Methods S2 ).
For immunoprecipitation, cell lysates were each divided into two equal concentrations and immunoprecipitated with anti‐TFEB or anti‐14‐3‐3 and Dynabeads coated with sheep anti‐mouse IgG (Invitrogen). The immunoprecipitated proteins were analysed by Western blots.
For immunoprecipitation, cell lysates were each divided into two equal concentrations and immunoprecipitated with anti‐TFEB or anti‐14‐3‐3 and Dynabeads coated with sheep anti‐mouse IgG (Invitrogen). The immunoprecipitated proteins were analysed by Western blots.
4
Pulse-Chase Analysis of TGFBIp Secretion
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Normal and GCD2 corneal fibroblasts were plated onto 35-mm culture dishes and cultured for 24 h. Cells were pre-incubated with cysteine-free DMEM (Sigma-Aldrich) containing dialyzed 0.5% fetal bovine serum (Invitrogen). Cells were incubated (pulsed) with 0.3 mCi/mL [35S] cysteine (PerkinElmer Life and Analytical Sciences, Boston, MA, USA) in the same medium for 20 min. After labeling, the cells were washed three times with PBS and incubated (chased) with DMEM containing 10% FBS at 37°C for 0, 15, 30, 60, 120, 180, or 240 min. The medium was collected and centrifuged at 4,500 × g for 10 min at 4°C. The cells were harvested in PBS, lysed in 500 μL RIPA buffer containing protease inhibitors, and centrifuged at 10,000 × g for 10 min at 4°C. Cell lysates and media were each divided into two equal volumes and immunoprecipitated with anti-TGFBIp and Dynabeads coated with sheep anti-mouse IgG (Invitrogen Dynal). The immunoprecipitated proteins were separated by 10% Tris/Glycine SDS-PAGE. The gels were enhanced with ENHANCE (Amersham Pharmacia Biotech) and exposed to Kodak BioMax X-ray films (Kodak, Rochester, NY, USA) for 7 days at −80°C.
5
Western Blot Analysis of Signaling Proteins
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Proteins were separated by 8% SDS-PAGE and transferred to nitrocellulose transfer membranes. The blots were blocked with freshly prepared 5% nonfat milk in TBST for 1 h at room temperature. Then the blots were incubated at 4 °C overnight with primary antibodies. After washing with TBST, the blots were incubated with horseradish peroxidase-conjugated (HRP-conjugated) donkey anti-rabbit IgG or sheep anti-mouse IgG (Invitrogen, China) at room temperature for 1 h. ECL substrate (CLiNX, Shanghai, China) and ChemiScope Touch (CLiNX, Shanghai, China) were used for detecting HRP-conjugated antibodies. Primary antibodies including anti-P-AMPK (phospho S496), anti-P-S6K (phospho S424), and anti-P-AKT (Ser473) were brought from Abcam (China). Primary antibodies including anti-LC3, anti-P-ERK(Thr202/Tyr204), anti-PARP1, anti-vimentin, anti-SQSTM1/p62, anti-GAPDH, and anti-IGF2R(M6PR) were brought from Proteintech (China).
6
NK Cell Depletion for Neutralization Assays
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For the neutralization assays using NK cell- depleted PBMC, depletions were performed on cryopreserved PBMC using mouse anti-human CD16 and CD56 antibodies (Invitrogen, Carlsbad CA) and Dynabeads (M-280) coated with sheep anti-mouse IgG (Invitrogen, Carlsbad CA), as per the manufacturer’s instructions. The PBMC were then PHA stimulated and verification of NK cell depletion was performed using flow cytometry; >90% of NK cells were depleted.
7
Methylated DNA Immunoprecipitation and Microarray Analysis
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Methylated DNA immunoprecipitation (MeDIP) was performed as we previously described (Cheung et al, 2010 (link)). Genomic DNA (5 μg) of tumour/control samples were sonicated on ice to generate random fragments of 100–500 bp in size. Fragmented DNA was subsequently heated at 95 °C, snap-cooled on ice and then incubated with mouse anti-5-methylcytidine monoclonal antibody (anti-5mC, Eurogenetec, Seraing, Belgium) in IP buffer containing 10 mM sodium phosphate (pH 7.0), 140 mM sodium chloride and 0.05% Triton X-100 for 2 h at 4 °C with gentle shaking. Magnetic beads conjugated with sheep anti-mouse IgG (Invitrogen, Grand Island, NY, USA) were added to the IP mixture and incubated for another 2 h. After incubation, the beads were washed three times in IP buffer and then digested by 80 μg of proteinase-K for 3 h at 50 °C. DNA was extracted by phenol/chloroform and ethanol precipitation. Following MeDIP, DNA was amplified using Whole Genome Amplification Kit (Sigma-Aldrich, St Louis, MO, USA), biotin-labelled and hybridised to Human Tiling Array 2.0R Chips (Affymetrix, Santa Clara, CA, USA) according to the manufacturer's instruction. After overnight hybridisation at 45 °C, chips were washed and stained on the Affymetrix Fluidic Station 450 and scanned on GeneChip Scanner GCS3000 (Affymetrix). Technical replicate of MeDIP-chip procedure was performed using the same DNA sample.
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