3 replies to this topic

[robby]

Hi everyone,

I recently did WB on cell lysate to detect my target protein. Strangely, I got two bands very close to each other. The ratio of these two bands varied and I wonder whether the upper one is due to a post translational modification such as phosphorylation. Did anyone meet such a problem? What other modification can cause such a gel shift in SDS-PAGE? Looking forward for your answer!


Robby

[yael]

robby, on Feb 21 2009, 12:54 PM, said:

Hi everyone,

I recently did WB on cell lysate to detect my target protein. Strangely, I got two bands very close to each other. The ratio of these two bands varied and I wonder whether the upper one is due to a post translational modification such as phosphorylation. Did anyone meet such a problem? What other modification can cause such a gel shift in SDS-PAGE? Looking forward for your answer!


Robby

Hello Robby,
2 bands on WB can definately hint to post translational modifications.
You did not mention: was the lysate of activated cells? if so, did you run a control non-activated cell ysate in parallel (on the same gel), and did you compare between them?
Also, you may have 2 (or more) isoforms of this protein with similar but not identical MW.

Hope this helps

[scolix]

robby, on Feb 21 2009, 11:54 AM, said:

Hi everyone,

I recently did WB on cell lysate to detect my target protein. Strangely, I got two bands very close to each other. The ratio of these two bands varied and I wonder whether the upper one is due to a post translational modification such as phosphorylation. Did anyone meet such a problem? What other modification can cause such a gel shift in SDS-PAGE? Looking forward for your answer!


Robby

This band could very well be a background band.

Its possible. Also another isoform, or post translational modification.

[rkay447]

Although phosphorylation is the most common modification detected by SDS-PAGE, there are many others that can cause shifts as well (glycosylation, acetylation, alkylation).  Plus there's always the possibility of an alternative spliceoform.  However, the shift of proteins is commonly used to demonstrate specific cellular activities.  Specific proteins are known to shift in a specific manner during cell cycle progression so you can use this to demonstrate cell cycle progression (or arrest).  The activation of specific signalling pathways results in known shifts of certain proteins so you can use this to determine if a pathway has been altered (ie: activated/inhibited).  The best way to determine if one of your bands is due to a phosphorylation is to treat half your lysate with phosphatase.  Run the treated with the untreated.  If the two bands collapse into one with the treated, you know you have a phosphorylated form.  By the way, a phosphorylated protein does not always result in a slower migrating band and in some instances (although rare) will be the faster migrating band.  For example, treatment with phosphatase causes the lower band of MCM2 to disappear.