Native & SDS - TOTALLY CONFUSED,kindly help me (Jul/31/2008 )

Hello every one..

I have a doubt and i am totally confused .I have isolated a protein and i runed on a 12.5% Native gel ( silver stained ) and i can see 3 bands ,band patterns are like this
there are 2 bands which are close to each other and are nearer to well and 3rd band some were middle of the gel.
If i run the same protein both in Reducing condition and Non reducing condition i am able to see only two bands close to each other ( position wise these bands are little bit below the two bands which was seen on native gel) but the 3rd band which was seen on native is completely disapered in both reducing condition and non reducing condition.....

from this result i am totally confused and not able to conclude my result .....

can any one help me in this regard.

Thanks in advance.

alie.

Can you post photos of your gels?

hi minnie mouse,

sorry i am not able to upload the pic... i explain you clearly i have a protein mol weight of 66kda.i runned the native gel and i extracted the protein from the gel.

The gel extracted protein (66kda) was again reloaded on the SDS PAGE in two conditions

1. protein sample + beta mercaptoethanol (ie Reducing condition)
2. Only protein Sample (ie Non reducing condition)

in non reducing condition i got single band at 66kda.So no doubt, my protein is 66kda

but in Reducing condition i am getting 3 bands ie one at 66 kda ,second band ~68kda and third band at ~70kda
problem arises here 66kda protein after reducing it should show bands below 66kda but i am getting bands above 66kda.?????????

i hope you got my point.kindly help me in this regard.

thanking you,

alie.

Possible your mercaptoethanol is contaminated?

hi jorge,
i also thought their may be contamination so i did with fresh chemicals like mercaptoethanol,acralamyed,loading buffer,tris buffer,running buffer ,TEMED,etc. but i am getting same pattren.i have repeated expt thrice.Same result so i am confused???????

alie

Does the reverse happen - i.e. can you extract any of the higher molecular weight species and run them on a non-reducing gel and see what they run as?

Do you have access to some gel filtration matrix? You could see if the 66kDa band is a single species if you have enough sample.
Are there antibodies raised against your protein? If so you could run a Western blot to see if the other proteins are non-specific (or if you have the facilities you could always extract the bands and wither N-terminally sequence them or get them analysed by Mass Spectrometry).

hi chris,

i have extracted the band from reducing gel and runned on non reducing.On nonreducing gel i am geting single band at 66kda.


i need to raise the antibody,so i am trying hard to clarify about this protein.and about MS we dont have that facility.

i dnt know about gel filtration matrix? can you tell me more on that.

alie.

chris sorry i know about gel filtration,i got confused about some other thing.....i have done gel filtration from that i am getting 3 bands.which are close to each other. so i followed electro elution.

alie.