Pupal brains and HzAm1 cells were lysed in NP-40 cell lysis buffer, and 500 μg of protein extract was used for coimmunoprecipitation. The coimmunoprecipitation systems contained 35 μl Protein G plus/Protein A-agarose suspensions (Merck Millipore, 3418211) and 1 μg V5-tag antibody (Merck Millipore, 3286106), 1 μg GFP antibody (Sangon Biotech, D110008), 1 μg JNK or FoxO antibody. The same amount of normal rabbit serum was used instead of antibodies as a negative control. Immunoblotting was performed with the corresponding antibodies, followed by incubation with Clean-blot HRP (Thermo Fisher Scientific, UB280382) 1:1000, and then the blot was detected.
V5 tag antibody
Manufactured by Merck Group
The V5-tag antibody is a laboratory reagent used to detect and purify proteins with a V5 epitope tag. It is a monoclonal antibody that specifically binds to the V5 peptide sequence, which is commonly used as a protein tag in recombinant protein expression systems. The V5-tag antibody can be used in various applications, such as Western blotting, immunoprecipitation, and affinity purification.
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2 protocols using v5 tag antibody
1
Coimmunoprecipitation of Pupal Proteins
2
Co-Immunoprecipitation of p-ERK and Myc-tag
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American cockroach appendages and KC cells from Drosophila embryo were lysed in NP-40 cell lysis buffer, and ~1000 μg of protein extract was used for co-IP. The co-IP systems contained 50 μl of Protein G plus/Protein A agarose suspension (Merck Millipore, 3418211), 1 μg of V5-tag antibody (Merck Millipore, 3286106), and 1 μg of p-ERK (Cell Signaling Technology, 4370S). The same amount of normal serum was used instead of antibodies as a negative control. Immunoblotting was performed with a myc-tag antibody (Cell Signaling Technology, 2276S) or CK-2 (Cell Signaling Technology, 2656S), after which the blots were incubated with Cleanblot HRP (Thermo Fisher Scientific, UB280382) at 1:1000, and then the blot was assessed.
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