7 replies to this topic

[Araz Zeyniyev]

Hi,

I am trying to purify hexahistidine tagged CcpA. When I run my induced E.coli cell-free extract on 15% PAGE (besides un-induced control of course) I see a clear overexpression but which is completely different from the size I expect.

Rumors:

-His6 makes proteins to run faster ! Though I doubt if 6 amino acids would make 10 kD difference.

-Cell-free extract were boiled 5-10 min 100C on hot-plate. Heating may lead to aberrat results.

Anybody has more scientific explanations on these?

Regards,
Araz

[Vini]

Hi AZ

6 -His will not make ur protein run faster....many a times u do observe such anomalous mobilities of proteins on PAGE........reasons may vary, from PTMs to hydrophobicity factors..........best way is to confirm the identity of ur protein either by W blot or mass spec or N-ter sequencing....

Best

Edited by DRN, 10 December 2009 - 10:55 PM.

[KAUSHIK THAKKAR]

Hi,

How much is the difference in the size?

How many times have you run the sample on gel? The reason why I am asking this, sometimes if there is some problem in gel percentage, such a thing might happen. I would suggest you to repeat the experiment, and see if you get the same thing...

If you get the same thing, then as suggested doing Western would be the golden standard.

Hope it works..!!


Let me know what happens.

Regards,
Kaushik

[KAUSHIK THAKKAR]

Sorry i did not see the topic of message, it mentioned about the size..

KAUSHIK THAKKAR, on Dec 29 2009, 12:21 PM, said:

Hi,

How much is the difference in the size?

How many times have you run the sample on gel? The reason why I am asking this, sometimes if there is some problem in gel percentage, such a thing might happen. I would suggest you to repeat the experiment, and see if you get the same thing...

If you get the same thing, then as suggested doing Western would be the golden standard.

Hope it works..!!


Let me know what happens.

Regards,
Kaushik

[Araz Zeyniyev]

Thank you to all.
Consensus sounds like sometimes gels just run unexpectedly. I am gonna repeat it. I am sure that protein is what is intended because it was previously isolated and characterised. I just need to make a new batch of pure protein and do some new experiments.
I am quite new in protein work so was quite surprised to see such a discrepancy.
I like DNAs more, thery always run as you expect

I'll let you know what happened.

Cheers,
Araz

[madrius1]

Keep in mind that some proteins run faster/slower than they are expected to. Have you ever seen this protein migrate at its expected size?

[blotted]

keep in mind that ecoli protein expression does not add postranslational modifications such us glycosilation which may change the running of your protein. Mammalian and yeast expression produce proteins glycosilated.

[HomeBrew]

Also keep in mind that accurate MW determination is difficult with a colored ladder -- the dye molecules change the apparent molecular weight of the bands, so they're difficult to compare to a native molecule.  Are you using a colored ladder?