Hi Guus,

By which method are you radio-labeling your DNA samples? If your DNA samples are also end-labelled, then it should be a matter of concentration or something wrong in your reaction. The problem is that if you radiolabel the marker internally by other methods such as nick-translation, bigger bands will show strong signals over lower ones. You solve a problem only to fall in another.

Another alternative is to have a longer exposition time for the marker. Cut it from the gel if necessary. Also, it is really nasty to work with radioactive agarose gel why you are not using polyacrylamide gel? They are easy to dry.

Hi Abdelhalim

thanks for your reply! My DNA samples are labelled with radioactive nucleotides and not end-labelled. The signal I get from that is pretty good, but my end-labelled marker is only visible after long exposure. As this is the final experiment before finishing up a manuscript I am trying to get a 'publication grade' image. So if there is any way to have both the marker and the sample in one image, without additional manipulation this would be great!

Hi Guus,

I don’t think this is an image manipulation only two exposure times and you should indicate clearly that to reviewers and in figure legend. Maybe you should indicate why you have chosen to perform 2 different ways of labeling. The only possibility is to end-label all and have longer exposure time for all. I think you could try that while you are finishing your manuscript. Good luck with it!

Hi Guus,

If you want two different exposicions in one image you can use two films. One film covers only the samples with have more signal and with the other film you cover the hole experiment. This way you´re shielding the radiactivity of your DNA samples and you can make longer exposures to both.

You can use more tan one film to make the shielding. Anyway, maybe you notice the limits of the two different films.

Good luck!

Hi Guus,

Why don't you cut and paste the two images from the longer and shorter exposures? Make sure they align perfectly. If you feel necessary, with an explanation to the editors (even to the readers in the legend) how the image was composed. It is perfectly legitimate in scientific publications.

Thank you all for your comments! I will proceed with the image analysis as suggested.

Hi Guus, Yanglong is right, most people never show their molecular weight markers in western blotting so I am sure that you could get your result and if required show the second figure as supplementary data. If yo do wish to get that one killer picture, why don't you measure the radioactivity sample as well as your ladder, and load the gel such that you are loading the same DPM in each band. That way they should all be about the same and your figure should be nice and neat. Good luck!

Hi Andrew,

There is no mean to achieve that unless he purifies each fragment individually. For the marker he has at most one 32P molecule per single molecule of the DNA ladder. The intensity of the signal depends only on the relative concentration of each band. For the DNA sample, the problem is a bit complicated if he has several bands. First, longer bands incorporate more 32P than shorter ones. Second, it will also depend on the relative frequency in each fragment of the dNTP that he has used for labeling.

Next time, it is better to end-label all. It doesn't matter if the exposition is a bit longer. Alternatively, there are several ways to increase the signal of the ladder.

1/He could subject the marker to both 5’-end labeling with PKN enzyme in the presence of gamma dATP and to 3’ extension with alpha dATP and Taq for example if the marker has blunt ends or a fill-in with klenow if it has cohesive ends. That would result in two fold increase in signal of the marker.

2/ Nick translation to internally label the marker or

3/ the best alternative is to synthesize own marker using radioactive PCR with defined fragment length.

4/ Of course, there might be more alternatives that I didn’t think about in this moment.

Hi Abdelhalim, by and large you are right, but my answer is really quite pragmatic so that Guus can just go the fridge/freezer and get his samples measure them in a scintilation counter and get a result today/tomorrow without buying any more labelling kits or 32P- I assume that Guss knows the number of bands he has and their size, so can calculate the rough fraction of the contribution of each band to the total dpm. The data from the commercial ladder is available-if it is a synthetic ladder then they will be weighted to give roughly the same amount of DNA per band, so they appear evenly loaded on an ethidium stained gel (THis generally maens that the smaller fragments will contain more ends). It is easier if it is a ladder based on a plasmid digest, as they should be all equally labelled. So using this information he can work out what the fraction of ends each band contains, and so can calculate the dpm needed to load so that the marker bands nearest in size to his experimental bands so when they are run on a gel will give roughly the same signal.