I am experiencing problems with one-dimensional Blue Native PAGE: It seems that proteins in the rage of 300 - 600 kDa are not properly transferred onto the PVDF membrane during Western Blotting of a BN-PAGE gel. Higher molecular weight proteins (>600 kDa) however seem to be transferred. I infer this from Ponceau staining and antibody staining, where the region of 300 - 600 kDa appears completely 'blotched out'.
Please see the attached file to get a better idea of what I mean.
I haven't done a Coomassie staining of the gel itself to check for the presence of protein in that region of the gel.
Does anyone have an idea what might be happening or has anyone observed a similar pattern while doing BN-PAGE?
The conditions that were used are the following:
6% gel (without SDS)
80 ug whole cell lysate per lane
transfer at 4 degrees at 30 V for 16 h, no MeOH in transfer buffer
Hi, Norbert,
it looks like you have "overtransfer2.. You can try to reduce either voltage or time. You also must be sure bthat your original gel isvOK, ea. try to staing gel with Coomasie blue. What about transfer of the marker?
Is it OK?
Hi Taras,
thank you for your advice. The marker tranfers alright (you can see it in the first lane), so the problem seems to be confined to the cell lysate. As I have just setting this technique up for our lab, I haven't played around a lot with different conditions.
You are right, it is worthwhile adjusting voltage and time. Do you mean by 'overtransfer' that the protein went straight through the membrane generating these 'blotches'?
Thanks, Norbert,
yes, as overtransfer I mean that protein went through the mebrane.
As for me, 30V and 16 h is a little bit long period. However, this also dependent form geometry of your membrane (area). There is a formula for calculation these parametrs, but better if you adjust this parametrs experimentaly.
I am not sure about the problem with cell lysate: if you have nice proteins with high MW, why should other protein pocess degradation?.
Hi Norbert,
but your overall transfer throughout all sizes looks quite promising, its just these "blotches" at a particular size that disturbes your nice transfer.
I my opinion there is "something" disturbing your running performance within the gel (loading buffer ingredient ect.) which is just in the samples not in the marker(!) and disturbes that particular sized band of "blotches" throughout your gel.
I would guess its something in the buffer of your samples (lysis buffer) or the loading buffer.
Sorry I cannot be more specific.
cheers Sven
I completely agree with Sven, except that the interfering component might as well come from the cell lysates. It can be a lipid or another biomolecule that gets transferred first and "blocks" the membrane not allowing proteins to bind. You can test this by attempting blocking the band with BSA and then staining it with Panceau.
As for the decision, changing voltage conditions might cause the band to migrate at a different level.
Alternatively, adding 0.01%SDS to your trasfer buffer might help to eliminate the band.
Thanks, Dimitri. I might try changing the running conditions and adding SDS to the transfer buffer first. If that doesn't work, I can still play around with the lysis buffer etc. That said, I already tried to include 0.1% SDS before, which almost completely inhibited the transfer itself (hardly any protein on the membrane detectable). Possibly, I was just too generous with the amount of SDS (0.1% instead of 0.01%)
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