22 replies to this topic

[leekaming]

There is a paper talking about what you have mentioned:

FEBS Lett. 1984 May 7;170(1):81-4.
SDS-PAGE strongly overestimates the molecular masses of the neurofilament proteins.

I also have situation similar to you. My protein is about 12kDa, but it appears as about 16kDa in SDS PAGE. My protein has a flexible and very negatively charged C-terminal tail, so i think the tail may hinder the mobility. The negative charge residues may also affect SDS binding to protein, thus lower the mobility.

[somu]

U can also try 130 V for 2 hrs or till the dye elute. What is your gel percentage

[MaggieRoara]

when i run my SDS-PAGE, it seems that themarkers don't fully separate out [there are meant to be 7 marker bands spread evenly, but only 4 appear, spread out with wide gapd between]...... does this mean that the smaller bands are not separating out? is this due to running conditions [200V for 1 hour)

cheers

is your gel heating up? try running at constant mAmps or lower the voltage

[M13]

when i run my SDS-PAGE, it seems that themarkers don't fully separate out [there are meant to be 7 marker bands spread evenly, but only 4 appear, spread out with wide gapd between]...... does this mean that the smaller bands are not separating out? is this due to running conditions [200V for 1 hour)

cheers

there might be possibility that the height of stacking gel is playing some role here. If the height is less than 1 cm than it can result poor resolution of protein markers.

[biochemi]

Could anybody suggest the reason for this case: my protein actually 30kDa but on SDS PAGE it migrate at 37kDa?

Is that the protein surface charge that involve in the migration of protein on SDS PAGE?

Thank you in advance.

hi
Check your protein may be glycated. In some cases glycated protein show such strange behaviour

[AntL]

Try a higher acrilamide concentration, or even better a gradient gel, with a lower voltage for longer time.

[MacXP]

I agree: It depends on the acrylamide concentration.
If you are using 6% gel, you won't be able to see those 10-20KD markers, or even 30KD markers if your gel is not that long.
You should choose wisely which concentration to use according to the molecular weight of the target protein.
The marker cannot run faster than the loading dye. But the chlorophyll can.

[@bhijit]

u can confirm protein by Western blot and forget about how it migrates.

Sometimes proteins dont migrate just based on their MW - protein characteristics affect the migration