Hi,
I did the Ponceau on the last run, and it came out looking like these two images, attached.
What could cause this, besides bad gel?
Thanks.
Another problem could be that in those samples (5th from right in 1st gel and 4th from the right in 2nd gel) there is a remaining genomic DNA. were those sample boiled as the other? and the protein concentration was equal?
Dear Aleks
Can you discusse your problems with more details.
I cannot understand your problems.
If your talking about the smearing effect in the fourth lane from the right, it looks to me like there was a piece of debris (possibly gel or cell pellet which you didnt separate out) which has run along with the sample on the lane.
Make sure you wash your wells before loading your samples, and clarify your samples well by removing all residual cells before loading.
Another problem could be that in those samples (5th from right in 1st gel and 4th from the right in 2nd gel) there is a remaining genomic DNA. were those sample boiled as the other? and the protein concentration was equal?
Hi Thank you for your response
Pedro, Rundk and Masood:
The 7th.lane on both membranes is much narrower than the other lanes. It looks better on the top membrane. On the 1st gel the lanes look over a lot better. They are much more even between each other
Fulvio, Vidya:
The samples were all prepared the same and then were normalized. Yes, the samples were boiled
The loading order is on both gels is exactly the same
The only difference is the 1st gel had 2 times less the loading amount.
The loading volume for both gels was exactly the same
I washed wells with buffer. I dont know a good way to do it. I just try to load it a few times with the running buffer, or pippet some of that out. I didnt notice a piece of gels there, you can usually see it as your sample wont load even, it would kind of load around it.
All the samples loaded even.
You can see that the lanes in the second gel are more uneven betweeen each other then the lanes on the 1st gel. Do you know what attributes to that
Thanks
It could be smeared proteins in this sample, and that could happend because of protein degredation or may be because of not enough boiling the protein so the proteins are not separated equally.
Ok, now seems a little more clear..so the total amount of protein is critical. In the 2nd gel proteins run more uneven because the gel is overloaded with protein so they run with difficulties. for the "strange" run of the 6th sample (from the marker) it could be that protein "stuck" together after boiling. Did you boil in normal loading buffer? Another suggestion..you extract those protein with a buffer-containing detergent? and then you dilute to final quantity in the same buffer? because you can try to dilute in detergent free buffer. Or if you dilute already in detergent-free buffer, you can try to do the opposite..In general terms, what you see here in 6th sample is protein aggregation that "pulls" (see the smear lines in 1st gel) , so you need to "break" those aggregate playing with detergents concentration
It may be due to bad or incompletely contact between paper and gel in the 7th line. If air buble existed, they prevent protein tranfer from gel to paper.
did you wash the membranes after putting the primary and secondary antibody???
I agree with Fulvio. We had this experience before and it was the genomic DNA contamination. Treatment with DNase could solve this problem. Did solve ours. Hope it does for you too!
It may be because of non similartity in volume or optimum concentration of protein or the length of duration of blotting and non accurate regulation of voltage or amper of electricity
You can get rid of DNA by sonicating the samples after adding SDS sample buffer and boiling. A sanitation bath is best-suited for this purpose. Sometimes it helps to reduce the protein concentration by adding more 1x loading buffer and then loading twice the amount of sample to each pocket. Also additional boiling/vortexing circles may help.
Aleks, high salt concentrations will indeed cause aberrations on your gel (and subsequently the transfer). You might also notice precipitates forming after you boil; I have faced this same problem when I prepare lysates in high salt buffers, which I could not afford to dilute because of the need to preserve phosphorylated residues on my protein(s) of interest.
Hi Aleks,
I'm having the same problem. How did you solve it? Today I tried more sonicating and DNAse I, but it didn't seem to improve.
Thanks
Hello,
I've been doing westerns for quite a long time but in the new lab with new RIPA buffer (Pierce one, not home made anymore), I started experiencing the same problem - bands just look as ugly as yours. With this buffer I am not able to drop down well debris and I have all this viscous genomic DNA floating around (before I used to have super solid pellet after centrifugation), so the tech guy told me to treat the samples with DNase as it contributes to bad running of my cell extracts. Will try it and tell you ASAP. Good luck!
I transferred protein from a 1D SDS-PAGE gel onto nitrocellulose membrane. low molecular weight Protein transfer ( from 66kda to 14 kda) )was succesful, but
high molecular weight protein didn't transfer at all...what can be the reason ?
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