Total cell lysates were generated in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40 (Genaxxon Bioscience), 10 mM EDTA, 100 mM PMSF, 200 mM DTT (Carl Roth), cOmplete (Roche), and phosphatase inhibitor cocktail 2 and 3 (1:100, Roche). Protein concentrations were measured via Bio-Rad protein assay (Bio-Rad). SDS-PAGE separated samples were transferred to nitrocellulose membrane. After blocking the membranes in 5% milk powder, membranes were incubated with human targeting primary antibodies goat anti-B7-H3 (CD276) (1:5000, R&D Systems), rabbit anti-AAMP (1:5000, Abcam), goat anti-GAPDH (1:1000, Linaris), mouse anti-α-Tubulin (1:500, Sigma-Aldrich), rabbit anti-β-Actin (1:1000, Cell Signaling). Secondary antibodies were horseradish peroxide-conjugated donkey anti-goat immunoglobulin G (IgG) (1:5000, Santa Cruz Biotechnology), sheep anti-mouse IgG (1:5000, Sigma-Aldrich), donkey anti-rabbit IgG (1:5000, Sigma-Aldrich), and mouse anti-rabbit light chain (1:10.000, Merck). Chemiluminescence development was performed using enhanced chemiluminescence (ECL) or ECL prime (Amersham) and visualized by developing Super RX-N films or ChemiDOC MP Imaging System (BioRad).
Sheep anti mouse igg
Manufactured by Merck Group
Sourced in United Kingdom
Sheep anti-mouse IgG is a laboratory reagent used in various immunoassay techniques. It is a polyclonal antibody produced in sheep that specifically binds to mouse immunoglobulin G (IgG) antibodies. This reagent can be used to detect and quantify mouse IgG in samples.
Automatically generated - may contain errors
Lab products found in correlation
Dithiothreitol (dtt), by Carl Roth (1 mentions)
Donkey anti rabbit igg, by Merck Group (1 mentions)
Rabbit anti aamp, by Abcam (1 mentions)
Ecl prime, by Cytiva (1 mentions)
Mouse anti α tubulin, by Merck Group (1 mentions)
Rabbit anti β actin, by Cell Signaling Technology (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Horseradish peroxidase hrp, by Merck Group (1 mentions)
L arabinose, by Merck Group (1 mentions)
Dab reagent, by Merck Group (1 mentions)
Protease inhibition cocktail tablets, by Roche (1 mentions)
Ecl plus, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Mahas4510, by Merck Group (1 mentions)
Goat anti mouse igg hrp, by Southern Biotech (1 mentions)
5 protocols using sheep anti mouse igg
1
Western Blot Analysis of Protein Targets
2
Recombinant Diabody Protein Expression
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Transformed E104 E. coli were propagated in Terrific Broth media (1% inoculum) to an O.D.600 of 2. At this time, the culture was induced with 0.1% l -arabinose (+) (Sigma) and incubated at 25 °C overnight. After centrifugation, supernatants were analyzed by Western blot to confirm the presence and size of the fusion proteins. Briefly, induced supernatants were loaded on a SDS-PAGE gel and transferred to a nitrocellulose membrane. Diabody and diabody fusion proteins were detected with an anti-Histidine (His) antibody (GE Healthcare, Buckinghamshire, UK) at a 1/1000 dilution followed by a sheep anti-mouse IgG coupled to horseradish peroxidase (HRP) (Sigma) at a 1/1000 dilution. The signal was developed using the DAB reagent (Sigma).
3
Antibody Selection for Prion Protein Analysis
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Proteinase K (PK) was purchased from Sigma (St. Louis, MO, USA). Protease inhibition cocktail tablets were from Roche Diagnostics (Indianapolis, IN, USA). Reagents for enhanced chemiluminescence (ECL Plus) were purchased from Thermo Scientific (Rockford, IL, USA). PrP-specific antibodies used in this study (Figure 2 ) included mouse monoclonal antibody mAb 3F4 against PrP106–112 [15 (link),39 (link)], mAb 1E4 against PrP97–105 [16 (link)], polyclonal antibody (pAb) Tohoku 2 (T2) against PrP97–103 [37 (link)], pAb EP1802Y against PrP217–226 [38 (link)], pAb anti-C antibody recognizing the C-terminal PrP220–231 [9 (link)], mAb 9A2 against PrP99–101 [40 (link)], mAb 12B2 against PrP90–94 [40 (link)], and pAb anti-N against PrP23–40 [41 (link)]. V14 and Bar209 mAbs recognize PrPC mono-glycosylated on the first (V14) (PrP168–181) or the second (Bar209) (PrP185–196) glycosylation sites, respectively, as well as unglycosylated forms [42 (link),53 (link),54 (link)]. Sheep anti-mouse IgG and donkey anti-rabbit IgG conjugated with horseradish peroxidase were purchased from Sigma Aldrich and used as secondary antibodies for the primary mAbs and pAbs, respectively [47 (link)].
4
LZ-8 Modulation of NF-κB Pathway
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LZ-8 was provided by Yeastern Biotech Co., Ltd., Taiwan. Anti-NF-κB (MAB3026), caspase-3 (AB1899), α-tubulin mAb (MAB1501) monoclonal antibodies (mAbs), horseradish peroxidase (HRP)-conjugated donkey anti-rabbit immunoglobulin G (IgG), and sheep anti-mouse IgG were all from Chemicon, Millipore, Temecula, CA. The nitrocellulose membrane (HybondTM-C Extra, Amersham Biosciences Corp., Hong Kong, China), enhanced chemiluminescence (ECL) Western blotting detection reagent and analysis system (WBLUC0100) were also purchased from Millipore, Temecula, CA. LZ-8 was dissolved in phosphate buffered saline (PBS) and stored at 4°C until the time of usage/application used.
5
Detection of antigen-specific antibody secreting cells
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Antibody secreting cells (ASCs) were assessed using ELISPOTs. Spleen (1 × 106 cells and 0.5 × 106 cells) or bone marrow cells (1 × 106 cells and 0.5 × 106 cells) were added to 96-well cellulose ester membrane plates (MAHAS4510; Millipore) coated with NP13-BSA (10 μg/mL) or GPI8-BSA (10 μg/mL) and incubated at 37°C and 5% CO2 for 8 h. To measure total IgG, wells were coated with sheep anti-mouse IgG (#AC111; Millipore) at 10 μg/mL. Concentration of cells to measure total IgG ASCs were 1 × 105, 0.5 × 105, and 0.1 × 105 per well. Anti-NP IgG, anti-GPI IgG or total IgG ASCs were detected using goat anti-mouse IgG conjugated to ALP (#3310-4; MabTek) and visualized using the BCIP/NBT system (# 50-81-00; KPL, USA). Spots were counted using an automated reader (AID ELISPOT Reader System, software version 7) and calculated as ASCs/1 × 106.
Blood was collected and serum separated at times indicated for ELISA. 96-well MaxiSorp plates were coated with GPI-BSA (10 ng/well) and titrated serum was incubated for at least 24 h at 4°C. Anti-GPI IgG was detected using goat anti-mouse IgG-HRP (SouthernBiotech) and visualized with 3,3′,5,5′- tetramethylbenzidine (TMB) substrate (#T8665, Sigma Aldrich). Antibody concentrations were quantitated using standard curves of known IgG protein concentrations.
Blood was collected and serum separated at times indicated for ELISA. 96-well MaxiSorp plates were coated with GPI-BSA (10 ng/well) and titrated serum was incubated for at least 24 h at 4°C. Anti-GPI IgG was detected using goat anti-mouse IgG-HRP (SouthernBiotech) and visualized with 3,3′,5,5′- tetramethylbenzidine (TMB) substrate (#T8665, Sigma Aldrich). Antibody concentrations were quantitated using standard curves of known IgG protein concentrations.
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