3 replies to this topic

[Meg P. Anula]

Hi, I am trying to express a HisTag recombinant protein using BL21(De3).

 

I'd normally grow and induce like 100ml bacteria culture, pellet, then lyse in probably 10ml of lysis buffer. Then sonicate and spin down the cells, which gives me like 10ml supernatant containing the crude protein. From there we'd use HisTag purification kit to isolate the target protein.

 

I believe that is the standard protocol.

 

Now I am online researching a good HisTag protein purification kit, and found that most of the minipreps are with each column fits only like 750ul clear lysates.  

 

That makes me wonder why would people purify from such small volume of lysates? How much induced culture (in ml) they used then? Is it enough for the eluted protein to be detected in western blotting? 

 

[Missle]

I have used mini-spin HisTag purification (homemade, not a kit but same principle).  I induce a 50mL culture and lyse in 1.25mL of lysis buffer.  I've lysed both by sonication and via SoluLyse.  Typically, I only purify half of that lysate and that is more than enough for a western blot - with adequate expression of course

[Meg P. Anula]

I have used mini-spin HisTag purification (homemade, not a kit but same principle).  I induce a 50mL culture and lyse in 1.25mL of lysis buffer.  I've lysed both by sonication and via SoluLyse.  Typically, I only purify half of that lysate and that is more than enough for a western blot - with adequate expression of course

 

 

Homemade? Meaning you bought the column but buffer are homemade? What brands?

[Missle]

Meaning that I purchased empty spin columns with frits and then added Nickel Sepharose to them.  I used my standard IMAC purification buffers.  I've made them from scratch before but I often buy GE's his-buffer kit because purchasing high-grade Imidazole that doesn't absorb at A280 is almost as expensive as buying the kit that includes it.