I did a western blot to detect MET of 145 KDa in cell trasfected with premiR of miR1, or premiR of miR133b, or in cell untrasfected or in cell trasfected with premiR scramble, I loaded the samples in the order: cell untrasfected, cell trasfected with scramble, cell trasfected withe premiR133b, cell untrasfected, cell trasfected with scramble and cell trasfected with PremiR-1.
I don't understand the result of actina the band that are located at the bottom because the bands are not all equal. What may be the problem other a problem of loading?
Thanks
Dear Franca,
The hollow bands you see are probably due to depletion of the substrate due to a very high amount of HRP. An indication of too much HRP can be that you may see brown/yellow bands on your membrane. HRP becomes brown when it is oxidized and inactive, and if you have a lot of HRP concentrated in one area, the proportion of inactive HRP will be visible to the naked eye. Also, too much HRP can produce free radicals in the presence of substrate, which can then inactivate HRP and lead to a loss of signal that would look similar to your membranes. I would suggest that you either load less protein on your gel, or use less primary Actin antibody - for example, a third or quarter of what you used for the above membrane. You could also try to use less secondary antibody.
I hope this helps,
Best wishes,
Roland.
Dear Franca,
The hollow bands you see are probably due to depletion of the substrate due to a very high amount of HRP. An indication of too much HRP can be that you may see brown/yellow bands on your membrane. HRP becomes brown when it is oxidized and inactive, and if you have a lot of HRP concentrated in one area, the proportion of inactive HRP will be visible to the naked eye. Also, too much HRP can produce free radicals in the presence of substrate, which can then inactivate HRP and lead to a loss of signal that would look similar to your membranes. I would suggest that you either load less protein on your gel, or use less primary Actin antibody - for example, a third or quarter of what you used for the above membrane. You could also try to use less secondary antibody.
I hope this helps,
Best wishes,
Roland.
I agree with Hjerpe, it looks like the depletion of substrate is the problem. If you had bubbles, you woud normally see some outlines of the bubble. Now, you can see white or grey 'ghost' bands of similar thickness. This actually indicates you have an even loading. You could try washing your current blot with TBST and apply the substrate again. Remember to work fast to prevent quenching of the substrate. You could try using chilled conditions to slow down the reaction. If that does not help, then load less protein as Hjerpe suggested.
I agree with Hjerpe and Sokka.
You can stripp your blot and re-blot against actin once more time, with lower concentration of primary and secondary antibodies. Instead, there is a lot of ECL products for chemiluminiscence detection. You can use a weak one for actin or instead dilute with water the one you have.
If you are using a machine for detection of the signal, you will need a good dynamic range in order to analyze the blot, if needed (even if it is doesn't make much sense to quantify something you don't know whether fitting into linear range and don't have a standard curve). If you are using autoradiography films and developer, use different exposition times. But too much signal is going to make this kind of things on your blots.
Dear Franca,
I totally agree with people stating that the substrate depletion is the root of your trouble. Try to use less protein and dilute your primary antibodies two- of four-fold. Also you can look at the membrane after ECL application and check for those weak yellow or brown bands Roland Hjerpe wrote about. I've noticed that intensity of these bands correlates with the actual amount of protein so they can be used for preliminary evaluation of protein amount (so you can understand if you need to adjust the amount of all samples or any of them in particular).
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