4 replies to this topic

[littlebrownbat]

Hi,
I'm quite new at protein work, I hope these questions aren't too "stupid"
Actually i just started western blot on my his-tagged recombinant proteins. Using anti-his antibody on my cell lysate (from transformed bacteria) yields ~4 bands (the 1st lane from left). I guess i must be having truncated proteins. However, I then transfected my HEK 293T cells to produce the same his-tagged protein to see if it's truncated as well, the bands that appear seem to resemble an sds-page run! u can see in my picture the 3 right most lanes. i can't figure out what's the problem.


I block with 3-5% BSA in TBST and use anti-his Ab at 1:10,000 (it's already conjugated to AP so i don't need a secondary Ab). I treat my samples in 10%SDS/DTT.

Also, it's very hard to find skim milk powder in supermarkers here and the one we have now is stated 'high calcium'. I've been  told that it will interfere with binding. May i know the reason why normal milk powder or full cream will not work? Will the fats interfere with blocking?

Thanks very much:D

Edited by littlebrownbat, 31 May 2011 - 12:57 AM.

[protolder]

littlebrownbat, on 31 May 2011 - 12:57 AM, said:

Hi,
I'm quite new at protein work, I hope these questions aren't too "stupid"
Actually i just started western blot on my his-tagged recombinant proteins. Using anti-his antibody on my cell lysate (from transformed bacteria) yields ~4 bands (the 1st lane from left). I guess i must be having truncated proteins. However, I then transfected my HEK 293T cells to produce the same his-tagged protein to see if it's truncated as well, the bands that appear seem to resemble an sds-page run! u can see in my picture the 3 right most lanes. i can't figure out what's the problem.
Photo-0606.jpg

I block with 3-5% BSA in TBST and use anti-his Ab at 1:10,000 (it's already conjugated to AP so i don't need a secondary Ab). I treat my samples in 10%SDS/DTT.

Also, it's very hard to find skim milk powder in supermarkers here and the one we have now is stated 'high calcium'. I've been  told that it will interfere with binding. May i know the reason why normal milk powder or full cream will not work? Will the fats interfere with blocking?

Thanks very much:D

Hola, for me the problem is inespecific interactions with some His.The cause  could be the imidazole concentration in washing buffer, you could increase a bit and second the proportion resin/ extract, if it is too high there are actives sites to bound inespecific His, no necessary 6His. Check the possibility of multimers of your band too, if you do the elution by a imidazole blow. About milk I haven´t any answer but it´s extrange , look in other supermarket, it´s a common product.  Buena suerte

Edited by protolder, 01 June 2011 - 10:04 PM.

[silkworm]

Do you have a positive control for the WB ( a purified His-tagged protein in buffer, and spike in untransfected 293T cell lysate)?

Also, you should use untransfected 293T cell lysate as negative control. The positive and negative controls can help you eliminate the possiblity to false positive/artifact signals.

[littlebrownbat]

Thanks protolder and silkworm, much appreciated

Yep, I included the untransfected 293T as a negative control, but I couldn't find differences in the lysate of the transfected and untransfected cells. Because we don't have the purified protein yet, I used transformed Rosetta as a positive control, WB detected that (left most lane in pic). I'm wondering if 293T cells have alot of his-containing proteins that's why my WB has so many bands?

Also, the other thing i'm thinking is that my transfection rate was low, hence there's no unique bands, maybe i should select for my transfected cells first before i do another sds-page.

[silkworm]

Hi,

Not sure the positive control ( transformed E.coli lysate ) is a valid one. You have to be sure those 4 bands are your proteins. Positive control for anti-His WB detection is better to be a His-fusion protein with known loading amount, MW. Transformed E.coli needs induction to express protein ( sometimes there's un-induced expression called leakage, no so commmmon).

My impression is your AP detection reagents created an artifact. Most if not all bands are non-specific. No evidence showed your protein is expressed in E.coli or 293T cells.

I'll get  a good positive control and retry WB with a new batch of AP-detection reagents.