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New Member
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Apr 1, 2007, 05:52 AM
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Protein phosphorylation
When using SDS-PAGE as a way of separating out protein of different sizes. If I have both phosphorylated Akt 2 and unphosphorylated Akt 2. Which will move the farthest through the gel? I know that by phosphorylating a protein you increase its mass by a small amount but if you phosphorylate a protein doesn't that increase its negative charge? Meaning that it would move further away from the cationic loading point in the gel? Please help I am very confused.
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Full Member
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Apr 1, 2007, 06:27 AM
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Debbie,
According to my knowledge, the phosphorylation of a protein does not affect its mobility in SDS-PAGE much and the reasons are these:
1) SDS covers each denatured protein with a strong negative charge and the negativity of PO4 is not enough to make a big difference in this charge.
2) PO4 also does not affect the protein mass or size by enough to make a significant difference in motility.
So, since the whole point of SDS-PAGE is to give proteins a negative charge proportional to their masses so that they will separate according to size... AND phosphorylation affects neither the charge nor the mass, of a protein, to make a significant difference, phosphorylation will not affect the migration of a protein on SDS-PAGE by much.
Hope this helps.
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New Member
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Apr 1, 2007, 08:58 AM
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 Originally Posted by manimuth
debbie,
According to my knowledge, the phosphorylation of a protein does not affect its mobility in SDS-PAGE much and the reasons are these:
1) SDS covers each denatured protein with a strong negative charge and the negativity of PO4 is not enough to make a big difference in this charge.
2) PO4 also does not affect the protein mass or size by enough to make a significant difference in motility.
So, since the whole point of SDS-PAGE is to give proteins a negative charge proportional to their masses so that they will seperate according to size....AND phosphorylation affects neither the charge nor the mass, of a protein, to make a significant difference, phosphorylation will not affect the migration of a protein on SDS-PAGE by much.
Hope this helps.
Hi Mani,
THANKS! It does, however the question that has been posed to me on this particular assignment is based on there being only two proteins in the lane. That of phosphorylated Act2 and unphosphorylated Act2 how would I tell the differnece between the two? The second part of the question asks how treating with phosphatase would affect the banding... now I know all that this does is dephosphorylates the protein. So if phophorylating the protein in the first place does not make any diference than treating it with phosphatase also will not make any difference...
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Full Member
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Apr 1, 2007, 02:46 PM
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 Originally Posted by debbie1504
That of phosphorylated Act2 and unphosphorylated Act2 how would I tell the differnece between the two? The second part of the question asks how treating with phosphatase would affect the banding...
Honestly, debbie, I don't think there will be a big difference. The phosphorylated protein might travel a bit slower. And treating with phosphatase should show two bands for two identical proteins (UNLESS, the phosphatase was not fully washed out, causing impurities and messing up the sample. But, I think the question probably implies good purification after treating with phosphatase.)
This is only my educated answer and if I am wrong, I hope someone will catch it. Please post if you find anything contrary. Thanks :)
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Ultra Member
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Apr 3, 2007, 11:49 AM
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Would you be allowed to do a northern blot and then use antibodies to ident the phosphorylated bands? That is the only way I can think to tell the difference.
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Junior Member
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Apr 4, 2007, 06:01 PM
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I've done these assays a few times, and my recollection is that phosphorylation usually causes a visible reduction in migration (i.e. band appears larger) on SDS-PAGE. Phosphorylation affects the binding of the SDS, giving the protein less total charge and thereby reducing mobility.
And generally, anti-phosphoprotein antibodies stink (at least they did 5-10 years ago when I was doing this assay), so a Western blot won't help you. Treating with phosphorylases will collapse the 2 bands back into one.
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