Any one currently working in the field of insect cell expression and recombinant protein purification (sf21 cells).
Not currently but I used to work on sf9 cell expression years ago. Anything I can help?
Thank you, My objective is expression and purification of my protein in sf21 cells. currently i am working with sf21 cells. I am new to this work and i prepared a construct using pIEXBac-3 vector from invitrogen. I tried for transfection and after detection of recombinant protein is very tough. I dont have specific antibody for that protein and also i need to use His tag or strep tag antibody for detection. Do you know any easy method for detection please let me know
As you said, you can detect your protein by western blot with anti-His or strep Ab.
Hello Sarathbabu,
I work with SF9 and SF21 cells for two years now. You used both Tags available in pIEX-Bac3, am I right?
The easiest way is to make a small-scale expression test (30mL) and taking a sample of 5mL every day up to 5 days. The cells are centrifuged at 5000xg, 5 min. Then you make small cell digest with all samples, take the supernatant aside and add SDS-sample buffer. The cells are resuspended in normal buffer and SDS-sample buffer and all samples are loaded on an SDS-Gel.
Then you perform the westernblot as it normally is established in ayour lab and then you detect your protein with His-Antibody. If you expect your protein to be expressed in a small manner (e.G a membrane protein), then you perform it with a primary Anti-His antibody and a secondary antibody (this way is more sensitive)
If you expect your protein to be expressed in higher amounts, you can use a HRP-couples antidody.
I hope that was helpful.
Best regards
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