The following antibodies were used in this study at the dilutions indicated for western blot (WB) and immunofluorescence (IF): anti-HA (BioLegend, 16B12, WB: 1:2000, IF: 1:1000), anti-GAPDH (Proteintech, 60004-1, WB: 1:5000), anti-PDCD7 (Proteintech, 12485-1-AP, WB: 1:500), anti-RBM41 (Atlas Antibodies, HPA042881, WB: 1:1000, IF: 1:200), anti-RNPC3 (Santa Cruz, sc-514951, WB: 1:500, IF: 1:100), anti-Sm (Invitrogen, MA5-13449, WB: 1:500), anti-SNRNP48 (Proteintech, 24297–1-AP, WB: 1:500), anti-V5 (Invitrogen, R960-25, WB: 1:5000), anti-ZCRB1 (Bethyl Laboratories, A304-697A, WB: 1:2000), Goat anti-Rabbit Alexa Fluor 488 (Thermo Fischer, A11008, IF: 1:500), Goat anti-Mouse Alexa Fluor 568 (Abcam, Ab175473, IF: 1:500), Goat Anti-Mouse IgG HRP, light chain-specific (Jackson ImmunoResearch, 115–035-174, WB: 1:10 000), Goat Anti-Mouse IgG HRP (Millipore, AP308P, WB: 1:10000), Donkey Anti-Rabbit IgG HRP (Amersham, NA934V, WB: 1:10 000). Mouse IgG2a kappa Isotype Control (Invitrogen, 14-4724-82) served as a negative control antibody in IP experiments. eBiocience Avidin-HRP (Invitrogen, 18-4100, WB: 1:500) was used to detect biotinylated proteins. Protein G-HRP (Millipore, 18-161, WB: 1:5000) was used in western blotting after IP to avoid detection of heavy and light chains from the IP antibody.
Mouse igg2a kappa isotype control
Manufactured by Thermo Fisher Scientific
The Mouse IgG2a kappa Isotype Control is a laboratory reagent used in flow cytometry and other immunoassays to establish the specificity of antibody binding. It serves as a control to differentiate between specific and non-specific antibody binding.
Automatically generated - may contain errors
Lab products found in correlation
Anti v5, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse alexafluor568, by Abcam (1 mentions)
Donkey anti rabbit igg hrp, by Cytiva (1 mentions)
Goat anti mouse igg hrp, by Jackson ImmunoResearch (1 mentions)
Goat anti mouse igg hrp, by Merck Group (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Anti ha, by BioLegend (1 mentions)
Transcription factor buffer set, by BD (1 mentions)
Mouse igg1 kappa isotype control, by Thermo Fisher Scientific (1 mentions)
Facsaria 2, by BD (1 mentions)
2 protocols using mouse igg2a kappa isotype control
1
Antibody Characterization for Western Blot and IF
2
Intracellular Profiling of Organoid Differentiation
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FACS intracellular staining: Organoids during the 2 nd ,5 th ,9 th and 14 th week of differentiation were dissociated into single cells by incubating them till dissociating at 37 o C within papain solution under shaking conditions. Then, the cells pelleted at 400xg for 5min and followed incubation with TryplE Select for 10min to ensure dissociation into singe cells. Further, the cells passed through 70μM
(2 nd week) -100μM (5 th , 9 th , 14t h week) cell strainer to avoid aggregates. For both digesting steps 10% FBS/DMEM-F12 as digesting deactivation solution applied to the cells. Then, for intracellular flow cytometric staining analysis the Transcription Factor Buffer set (BD Pharmigen) was applied and the cells were analyzed using flow cytometer (BD Biosciences FACS ARIAII).
Primary antibodies used in this study: anti-PAX7, anti-MYOD1, anti-Pax3 in total amount of 400μg per staining, while as secondaries the Rhodamine RedTM-X (RRX) AffiniPure Goat Anti-Mouse IgG, Fcγ Subclass 1 Specific (Jackson Immunoresearch Laboratories), Alexa Fluor® 488 AffiniPure Goat Anti-Mouse IgG, Fcγ subclass 2a specific (Jackson Immunoresearch Laboratories) in 1:50 dilution. As isotype controls, Mouse IgG1 kappa Isotype Control (Invitrogen, clone P3.6.2.8.1), Mouse IgG2a kappa Isotype Control, (Invitrogen, clone eBM2a) were used at 400μg total amount per staining.
(2 nd week) -100μM (5 th , 9 th , 14t h week) cell strainer to avoid aggregates. For both digesting steps 10% FBS/DMEM-F12 as digesting deactivation solution applied to the cells. Then, for intracellular flow cytometric staining analysis the Transcription Factor Buffer set (BD Pharmigen) was applied and the cells were analyzed using flow cytometer (BD Biosciences FACS ARIAII).
Primary antibodies used in this study: anti-PAX7, anti-MYOD1, anti-Pax3 in total amount of 400μg per staining, while as secondaries the Rhodamine RedTM-X (RRX) AffiniPure Goat Anti-Mouse IgG, Fcγ Subclass 1 Specific (Jackson Immunoresearch Laboratories), Alexa Fluor® 488 AffiniPure Goat Anti-Mouse IgG, Fcγ subclass 2a specific (Jackson Immunoresearch Laboratories) in 1:50 dilution. As isotype controls, Mouse IgG1 kappa Isotype Control (Invitrogen, clone P3.6.2.8.1), Mouse IgG2a kappa Isotype Control, (Invitrogen, clone eBM2a) were used at 400μg total amount per staining.
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