Jag1 and Mib1 constructs were co-transfected into sub-confluent HEK293T cells in a 6-well plate using Lipofectamine 2000 (Invitrogen, Carlsbad CA USA). ΔRNG Mib1 was used for all experiments except Figure S1A , which includes additional Mib1 molecules as indicated. After 48 hr, cells were lysed on ice in 500 μL buffer containing 0.5% w/v Fos-Choline 12, 50 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM DTT, 5 μM ZnCl2, 1 mM CaCl2 and EDTA-free protease inhibitors (Roche, Basel SUI). Lysates were pelleted by centrifugation and the supernatant was incubated with α-V5 agarose (Sigma-Aldrich, St. Louis MO USA) at 4 C for 1 hr. Following incubation, agarose beads were washed 3x for 5 min each and then re-suspended in SDS-PAGE loading buffer. For Western blotting, α-Myc (clone 9B11, Cell Signaling, Danvers MA USA) and α-V5 (polyclonal ab9116, Abcam, Cambridge UK) antibodies were used in 5% w/v milk TBST blocking buffer.
α myc clone 9b11
Manufactured by Cell Signaling Technology
Sourced in United States
α-Myc (clone 9B11) is a monoclonal antibody that specifically recognizes the c-Myc protein. It is a useful tool for the detection and analysis of c-Myc expression in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Edta free protease inhibitor, by Roche (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Poly l lysine slides, by Avantor (1 mentions)
Axioscope microscope, by Zeiss (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
2 protocols using α myc clone 9b11
1
Mib1 and Jag1 Co-Transfection Assay
2
Immunofluorescent Localization of MYC in T. brucei
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bloodstream-form T. brucei (1 × 106) were fixed in 3% paraformaldehyde in PBS (Sigma) for 10 min at RT before washing twice with PBS and settling onto poly-L-lysine slides (VWR) for 1 h at RT. Cells were permeabilized with 0.5% Triton X-100 (Sigma) in PBS for 15 min and blocked with 50% FBS (Sigma) in PBS for 15 min, followed by incubation with primary α-MYC (clone 9B11, Cell Signalling) at 1:5,000 in 3% FBS in PBS for 1 h at RT. Finally, slides were incubated with α-mouse Alexa Fluor-488-coupled secondary antibody (ThermoFisher) at 1:2,000 for 1 h at RT before VECTASHIELD (Vector Laboratories) with DAPI (4′,6-diamidino-2-phenylindole) was applied and a coverslip was used to seal on top. Z-stacks were captured using a Zeiss Axioscope Microscope and merged using the ImageJ Maximum Intensity Projection algorithm.
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!