SDS polyacrylamide electrophoresis (PAGE) was performed using 8–16% gradient gels (Thermo Fisher Scientific, Dreieich, Germany). Colloidal Coomassie staining was performed using Imperial Protein Stain (Thermo Fisher Scientific, Dreieich, Germany). For pulldown, 293T cells were transfected with pcDNA3, pcDNA6aV5-KSHV-gH, or pcDNA6aV5-KSHV-gH-ASAELAAN and pcDNA6-KSHV-gL-Flag using PEI in a 1:10 ratio (gH/gL). Lysates of 293T cells transfected with the respective expression constructs for gH-V5/gL-Flag complexes and lysates from non-transfected cells were prepared in NP40 lysis buffer (1% Nonidet P40 Substitute (Sigma-Aldrich), 150 mM NaCl (Sigma-Aldrich, St. Louis, MO, USA), 50 mM HEPES pH 7.5 (VWR), 1 mM EDTA (Amresco, Solon, OH, USA) with freshly added Protease Inhibitor Cocktail (Amresco). Subsequently, lysates were incubated with 1 μg V5-tag antibody (BioRad, Feldkirchen, Germany) and ProteinG sepharose (GenScript, Piscataway, NJ, USA) overnight at 4 °C with agitation. ProteinG beads were collected by brief centrifugation and washed three times in NP40 lysis buffer. Precipitates were heated in 2× SDS sample buffer (95 °C, 5 min). Western blotting was performed as described previously [27 (link)] using the respective antibodies (Table 2 ).
Protein g sepharose
Manufactured by GenScript
Sourced in United States
Protein G Sepharose is a protein affinity chromatography resin used for the purification of immunoglobulins and other proteins. It consists of the bacterial protein G covalently coupled to Sepharose beads. Protein G has a high affinity for the Fc region of various immunoglobulin classes, allowing efficient capture and purification of antibodies from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Avantor (1 mentions)
Gradient gel, by Thermo Fisher Scientific (1 mentions)
Imperial protein stain, by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Avantor (1 mentions)
V5 tag antibody, by Bio-Rad (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Nonidet p40 substitute, by Merck Group (1 mentions)
Protein g sepharose beads, by GenScript (1 mentions)
Foxp3 staining kit, by Thermo Fisher Scientific (1 mentions)
Facs symphony, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
α cd11b, by BioLegend (1 mentions)
Pngase f, by New England Biolabs (1 mentions)
6 protocols using protein g sepharose
1
Protein Pulldown and Western Blot Analysis
2
Immunoprecipitation of Viral Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were transfected or infected as indicated for each experiment. Between 200 and 500 µg of proteins was precleared overnight at 4°C with protein G Sepharose beads (GenScript). Beads were removed by centrifugation, and supernatants were incubated with either anti-E4orf6 polyclonal antibody 1807, anti-E1A mouse monoclonal antibody, or anti-E2F1 KH95 mouse monoclonal antibody, followed by incubation with protein G Sepharose (GenScript). The beads were extensively washed in lysis buffer and examined by SDS-PAGE. The detection of Rb and HA-tagged proteins in Western blots with primary mouse monoclonal antibodies was done with secondary HRP-conjugated rat anti-mouse κ light-chain-specific antibody.
3
Flow Cytometric Analysis of Mouse Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies against mouse CD4 (clone GK1.5) and CD8α (clone YTS196.4) were purified from hybridoma culture supernatant using protein G Sepharose (Genscript, Piscataway, NJ). Antibodies against PD-1 (clone RMP1.14) were purchased from BioXCell. Tetrameric peptide-MHC complexes were generated and staining was performed as described previously [21 ]. Fluorochrome-coupled antibodies for flow cytometry were purchased from eBioscience, BioLegend, or BD. Single-cell suspensions and enriched tumor-infiltrating leukocytes were incubated for 10 min with 0.5 mg/ml anti-CD16/CD32 (clone 2.4G2, produced in house) to block Fc-receptors followed by staining with fluorochrome-coupled antibodies for 20 min at 4 °C in FACS buffer (PBS 1% BSA, 20 mM EDTA). FoxP3 was detected using a FoxP3 Staining Kit (eBioscience) in accordance with the manufacturer’s instructions. Erythrocytes were lysed using red cell lysis buffer (RCLB). Samples were analyzed on either a LSR-II, FACSCanto II, or a FACSSymphony (Becton Dickinson, Mountain View, CA) and data were analyzed with FlowJo Analysis Software (Tree Star Inc, Ashland, OR).
4
Antibody-Induced Murine Model of Oral Pemphigoid
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To generate IgG against the murine α3 chain of laminin 332 (mLAMα3), New Zealand white rabbits were immunized with a mixture of recombinant His-tagged fragments of middle and C-terminal mLAMα3 portions (11 (link)). Total rabbit IgG was affinity purified using protein G Sepharose (Genscript, Piscataway, NJ, USA). Reactivity of purified IgG was analyzed by indirect immunofluorescence (IF) microscopy on murine skin. B6 mice were injected every other day with 6 mg of rabbit anti-mLAMα3 IgG or normal rabbit (NR) IgG. B6 mice were selected for these experiments because they are so far the only strain used in this model (11 (link)). Randomization and treatments were performed as described for antibody transfer-induced EBA. Different body parts were individually scored by the appearance of crust, erythema, lesions, and alopecia. On Day 12, high-resolution endoscopy (HOPKINS Optik 64019BA; Karl StorzAidaVet, Tuttlingen, Germany) of mouth oral cavity (i.e., pharyngeal mucosa, tongue, and right/left buccal) was conducted to determine the extent of oral lesions. Blood and tissue samples were taken for further analysis. For oral score, we determined 1 score point for each affected area: tongue, left buccal mucosa, right buccal mucosa, pharynx.
5
Purification of Blocking Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blocking antibody against CD11b or FcγRIV (α-CD11b or α-FcγRIV) in PBS without sodium azide were purchased from Biolegend. 75 μL PNGase F (New England Biolabs, P0704L) was added to 5 mg α-CD11b or α- FcγRIV at 37 °C for 4 h. The antibodies were repurified over protein G-sepharose (Genscript).
6
Induction of Experimental Murine Laminin Alpha 3 Autoimmunity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The induction of experimental MMP has been described in detail previously (13 (link), 20 (link)). In brief, anti-murine laminin alpha 3 (mLama3) IgG was generated by immunization of New Zealand white rabbits with recombinant His-tagged fragments directed against the mid (amino acids 1656–1985) and C-terminal (amino acids 2756–3330) domains of mLama3. Total rabbit IgG was affinity purified using protein G Sepharose (Genscript, Piscataway, NJ, USA). C57BL/6J mice were injected s.c with 6 mg anti-mLama3 IgG or normal rabbit IgG every other day from Day 0 to Day 10 to induce disease. Experimental groups were distributed in a blinded manner and mixed within the cages.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!