Q: Will this kit detect EGFP?
A: The polyclonal antibodies used in the GFP ELISA kit (AKR-121) are against Jellyfish GFP (Aequorea Victoria) and will detect its variants EGFP, YFP, BFP and CFP, which are only point mutations of wild type GFP. The recombinant GFP standard used in the kit is also EGFP.
Q: After we have prepared our GFP lysate sample, can we freeze the lysate for later testing?
A: GFP is a very stable molecule. When preparing the lysate we recommend using lysis buffer containing proteinase inhibitors. Then you can freeze your sample at -20ºC for a minimum of 3 months, or for 1 year at -80ºC.
Q: What is the protocol for lysing cells?
A: Here is the protocol we recommend for lysing cells:
Wash cells once with cold PBS, then add cold 1X Assay/Lysis Buffer and incubate 5-10min at 4ºC. Collect lysate (supernatant) by centrifuging 10,000g for 5 minutes at 4ºC. Any detergent based lysis buffer, such as RIPA, is compatible with this assay.
Q: What lysis buffer do you use?
A: Any detergent based lysis buffer, such as RIPA, is compatible with this assay. Here is the lysis buffer we use: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 % Triton X-100 or 1 % Nonidet P-40, 1 mM EDTA, 1 mM EGTA, and Proteinase inhibitor cocktail.
Q: Can lysates be stored prior to using the assay?
A: GFP is a very stable molecule, so as long as your lysis buffer contains proteinase inhibitors the lysates can be stored at -20ºC for three months or at -80ºC for one year.