Chinese Hamster Ovary (CHO) cells were cultured in Alpha-MEM (Invitrogen Life Technologies, Carlsbad, CA) containing 10% FBS and 2 mM L-glutamine, at 37°C in a 5% CO2 incubator. The cells were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 10 min, and permeabilized with 0.1% Triton X-100 in PBS for 15 min. The cells were blocked with 1% BSA, 0.05% Tween 20 in PBS for 30 min, and incubated with rabbit anti-Clathrin heavy chain (Abcam, Cambridge, MA, 1∶500) antibody at 4°C overnight. ATTO 425 (Fluka, Buchs, Switzerland)-conjugated goat anti-rabbit IgG (Invitrogen Life Technologies, Carlsbad, CA) was used as the secondary antibody, and incubated with the cells for 1 hr. The cells were washed with PBS or 0.05% Tween 20 plus PBS for 5 min three times between each step. After washing with 0.1% BSA, 0.05% Tween 20 in PBS, The cells were mounted with mowiol (Sigma-Aldrich, Canada).
Rabbit anti clathrin heavy chain
Manufactured by Abcam
Sourced in United Kingdom
Rabbit anti-clathrin heavy chain is a primary antibody that recognizes the heavy chain of the clathrin protein. Clathrin is a major component of the clathrin-coated vesicles involved in endocytosis and intracellular trafficking. This antibody can be used to detect and study the expression and localization of clathrin heavy chain in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Alpha mem, by Thermo Fisher Scientific (1 mentions)
Mowiol, by Merck Group (1 mentions)
Goat anti rabbit igg af 647, by Thermo Fisher Scientific (1 mentions)
Prolong gold with dapi, by Thermo Fisher Scientific (1 mentions)
Rat anti mouse mhc 2 clone 2g9, by BD (1 mentions)
Lms 700 confocal microscope, by Zeiss (1 mentions)
Poly l lysine coated coverslips, by Corning (1 mentions)
Af555 goat anti rat igg, by Thermo Fisher Scientific (1 mentions)
Quick start bradford protein assay, by Bio-Rad (1 mentions)
Rat anti ha 3f10, by Roche (1 mentions)
Bmg fluostar omega, by BMG Labtech (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Nupage novex 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Rabbit anti gfp, by Abcam (1 mentions)
Mouse anti alix, by BD (1 mentions)
Mouse anti gapdh 6c5, by Abcam (1 mentions)
Mouse anti hsc hsp70 n27f3 4, by Enzo Life Sciences (1 mentions)
Mars data analysis software, by BMG Labtech (1 mentions)
Eclipse ti microscope, by Nikon (1 mentions)
4 protocols using rabbit anti clathrin heavy chain
1
Immunofluorescence Staining of Clathrin in CHO Cells
2
Immunofluorescent Staining of Mouse MHC-II
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Skin cell suspensions were obtained by Liberase digestion as mentioned above. Cell suspensions were diluted in RPMI and deposited in microplates containing poly-L-lysine-coated coverslips (Corning). Cells were incubated overnight at 4°C and fixed with 4% paraformaldehyde. Cells were then incubated with rat anti-mouse MHC-II (clone 2G9, BD Bioscience) and rabbit anti-clathrin heavy chain (Abcam), followed by AF647 goat anti-rabbit IgG (Invitrogen) and AF-555 goat anti-rat IgG (Invitrogen). Finally, coverslips were mounted on microscope slides using ProLong Gold with DAPI (Life Technologies). Cells were visualized with a LMS 700 confocal microscope (Zeiss) and pictures were edited using Zen software (Zeiss).
3
Comprehensive Western Blot Protocol
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For Western blot analysis, protein concentrations were measured by Quick StartTM Bradford Protein assay (Bio-Rad) using the plate reader Fluostar Omega BMG (BMG Labtech) and the corresponding MARS Data Analysis Software (BMG Labtech). Proteins were separated on NuPAGE®Novex® 4–12 % Bis-Tris Protein Gels (Life Technologies) followed by transfer onto a PVDF membrane (GE Healthcare) in a wet blotting chamber. Western blot analysis was performed using mouse anti-LDL receptor (1:500; NovusBiologicals); mouse anti-Alix (1:1000; BD Bioscience); rat anti-HA 3F10 (1:1000; Roche); mouse anti-GAPDH 6C5 (1:5000; Abcam); mouse anti-Hsc/Hsp70 N27F3-4 (1:1000; ENZO); mouse anti-VSV-G 5D4 (1:1000; Sigma); rat anti Sup35 M domain (1:10; hybridoma supernatant);88 (link) rabbit anti-Tau ab64193 (1:1000; Abcam); rabbit anti-Flotillin 1 ab133497 (1:1000; Abcam); mouse anti-SARS-CoV-2-spike S GTX632604 (1:1000; GeneTex); rabbit anti-hACE2 ab15348 (1:1000; Abcam); rabbit anti-clathrin heavy chain (1:1000; Abcam); rabbit anti-GFP (1:5000; Abcam). The membrane was incubated with PierceTM ECL Western Blotting Substrate (Thermo Fisher Scientific) according to the manufacturer´s recommendations and imaged with the Imaging system Fusion FX (Vilber Lourmat).
4
Visualizing Clathrin and BioID2 in HEK 293 Cells
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HEK 293 and Flp-In™ T-REx™ 293-BioID2-GFP1–10 stable cells were transfected with GFP expression plasmids as indicated in the text. Flp-In™ T-REx™ 293-BioID2-GFP1–10 stable cells were incubated in the presence of tetracycline to induce BioID2-GFP1–10 expression. Green fluorescence in live cells was imaged using a Zeiss Axio Observer inverted fluorescence microscope equipped with an Axiocam 506 mono camera.
To localize clathrin and BioID2, HEK 293 cells were transfected as described above. On the following day, cells were fixed with 4% paraformaldehyde and permeablilized with 1% Triton X-100. Clathrin was detected by sequentially probing with rabbit anti-clathrin heavy Chain (Abcam, Cambridge, UK, product number ab21679) primary antibodies and Alexa Fluor 594 goat anti-rabbit IgG secondary antibodies (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA, product number A11012). BioID2 was detected with chicken anti-BioID2 primary antibody and Alexa Fluor 488 goat anti-chicken IgG secondary antibodies (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA, product number A11039). Cells imaging was performed using a Nikon A1 Confocal System on Nikon Eclipse Ti Microscope.
To localize clathrin and BioID2, HEK 293 cells were transfected as described above. On the following day, cells were fixed with 4% paraformaldehyde and permeablilized with 1% Triton X-100. Clathrin was detected by sequentially probing with rabbit anti-clathrin heavy Chain (Abcam, Cambridge, UK, product number ab21679) primary antibodies and Alexa Fluor 594 goat anti-rabbit IgG secondary antibodies (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA, product number A11012). BioID2 was detected with chicken anti-BioID2 primary antibody and Alexa Fluor 488 goat anti-chicken IgG secondary antibodies (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA, product number A11039). Cells imaging was performed using a Nikon A1 Confocal System on Nikon Eclipse Ti Microscope.
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