6 replies to this topic

[Curtis]

I'm trying to tag my gene, but I don't have good memories with FLAG since its antibody for purification (anti-FLAG M2) is very detergent sensitive. The manual says I can't use, Triton, Tween 20, CHAPS or digitonin, so its like I can't lyse my cells with any popular lysis buffer. I have never used His plasmids before, and I want to know if its purification antibodies are as sensitive as FLAG's? The other thing is that I want to use a purification column this time instead of beads/affinity gel, just to practice. SIGMA does not have any column for FLAG, but His columns are available everywhere. which one to buy?

[bob1]

My protein purification colleague says to steer away from his and look at GST tags instead; he says they are much more soluble and more specific than his.

[Curtis]

thanks Bob

[PhDinAcronyms]

I agree with bob1, GST tags are more specific and usually easier to purify in one step. They're also more soluble but sometimes have problems of their own. I've gotten His tagged proteins quite clean once you establish a good protocol, usually washing with gradients of imidazole. I didn't use his antibodies though, you can use nickel sepharose to purify and then wash slowly with an increasing amount of imadazole to get rid of the few non-specific proteins that show up before eluting.

[Curtis]

Yes, that's one of the advantages of His. You can purify without antibody, but FLAg definitely needs antibody M1. M2 or M5, and they seem to be detergent sensitive. I read an article that compared many tag systems and FLAG was the most expensive method.

I'm not sure what GST vector Bob's friend is using. Most of the vectors I have found so far are around 5 kb. Now, my gene of interest is around 6.6 kb and this will make a huge plasmid together. I'm not sure if His-tag is suitable for long fragments, or GST is more efficient for a 6.6 kb gene?

In addition, I want to tag the c-terminal, but most GST vectors are designed for n-terminal tagging.

[Adrian K]

Do you have a chromatography system? you can use it with column to purify his-tagged protein using gradient / step-wise elution, and do another round of ion exchange to further purify your protein.

I can give you some of my contact of suppliers if you are still in Malaysia, just pm me.

[Curtis]

My friends also have some Invitrogen columns I can borrow. Thanks Adrian.