just a few thoughts:

- maybe you do not add enough probe in your assays or you add too much cell extracts or proteins to be tested. Can you perform a titration gel with dilution of your extracts or proteins ?

- how did you label your probe ? usually for radioactive probe, you labell one strand (oligo), remove free nucleotide and anneal with an 10x excess of cold probe

- are you expecting a competition with your mutant probes ?

- High exposure of your gel, maybe you have a saturation.

The first explanation is the most likely. Good luck in your experiments.

Hi there,

I guess the probe lane correspond to the experiment made without protein, right?

Have you ever run a probe sample just to see how it behaves on the gel compared to your experiment control? It would definitely tell you if the problem is from the experiment or from the gel (or both...).

Running the gel for too long? Free-probes may go away 

Hello

Biotin-probes ? Are those purified ? If not, free biotin-labeled nts could bind to your protein. It looks linke your free probe is weakly label. Considering the fact that you put the same amount of material in each slot, the signals obtained are likely to be due to free biotinylated-nts.

Try to purifiy your probe after optimization of the incorporation.

If the probes correspond to purified material, the tests describe above should give you the correct running condition on your gel.

Best

To check the probe you can also made a dot blot. You can put different amounts of probe in a membrane and check the probe concentration and intensity of signal to see if everything is OK.

just a few thoughts:

- maybe you do not add enough probe in your assays or you add too much cell extracts or proteins to be tested. Can you perform a titration gel with dilution of your extracts or proteins ?

- how did you label your probe ? usually for radioactive probe, you labell one strand (oligo), remove free nucleotide and anneal with an 10x excess of cold probe

- are you expecting a competition with your mutant probes ?

- High exposure of your gel, maybe you have a saturation.

The first explanation is the most likely. Good luck in your experiments.

Dear all, before having Henry Lee checking all these, let him check the purity and the labeling efficiency of his probe first.

D

Thanks for all,

       I need to add some details.

      - I run my gel by 100v,4-5mA for an hour.The blue loding dyes are nearly in the middle of the gel,so I think it could not been over run.

      -The probes were labbelled by an experienced corporation. I only have to finished the annel reaction  after I recieved it. The probe was labelled by a kind of biotin in 5‘ side of each   strand. The concentration of the binding probe is 10fmol per ul and the cold/mutant competitor probe is 5nmol per ul. I got this by learning some articles which have been published by my own lab. I believe it is OK.

       -The protein is about 31ng per ul, I added 0.4ul. It is not too much either.

      -I will try to change some density in order to figure out something.

Have you put enough glycerol in the loading buffer in your probe?

Glycerol was added 1ul and 5Xloding buffer was added 5ul to the sample of which total volume is 25ul .

Look like you have got a band for the probe alone. Are you doing a competition assay? if that so does mutant probe 1 and 2 are supposed to compete with your real probe and the bound complex then should get smaller but I can see all the bound complex bands are huge. What elements are being labelled here, look like more than one. And how can you measure the band intensity? I need all these info to get to the answer.

Hi Henry,

  You may also need to have lanes for mutant probes without any protein

Hi Yue，

Thank you  for reminding  me of this.

But I still have to find out why there is no free probe bands first.I attached a new photo for reference.

I have  eliminated the possibility of running too long. And the amount of  binding probe and the protein are all suitable.The probes were told not to be purified because that have been done by the corporation.

Hi, perform the same experiment with a probe "purified" by gel filtration of on a gel (don't always trust corporation 'purification'...that can be a simple desalting step...). Also, to have some ideas of what happen here, you may need to put sf1 peptide alone as well to see if there is any interference with your revelation method and discard this hypothesis : "Does this peptide somehow binds streptavidine or the ligand used to reveal biotynilated material ?"

Your data with unlabeled competitor is strange. Which ratio label/unlabel has been used ?

Best

Hi David,

           I only have done this  expriment for less than 3 months, and I really don't  unstand the suggestion of  discarding the hypothesis which I know nothing about it.

           I will try another protein or run gel with different amount of probes wtihout any protein in able to learn somrthing.

           The ratio of label/unlabel is 1/500 as well as the ratio of label/mutant.

            In which way do you think the data is strange? No free probe at all? BUT the free  probe of other lanes are all wrong for being so thin.

Discarding the hypothesis : you need to make the test if SF1 peptide can somehow bind the reagent used for probe visualization (as seen in lane 3 vs 1 and 4, this is apparently not the problem since signals are distinct but ... if protein loading differs, that could explain why : test protein alone and reveal).

is SF1 splicing related or stands for something else ?

you did not mentioned the nature of the probe DNA, RNA, single stranded, double stranded ?

For your competition, the strange comment is that the binding of probe appears complete (no free band down in the gel) and low signal in the shift, whereas with mutant competitor some probe remains. I think you standardized the probe amounts but prefer to ask : Are the amounts of probe standardized for all lanes ?"

I suggest you using the condition Biot-probe + variable amount (diluted 1/5) of SF1 to determine the best condition for getting a "good signal", then apply this together with competitor/or unlabeled. Again, I suggest you to make your assay with purified probe.

Best

D

Hi, Henry

Maybe you can try Cy5.5 to label your probe (you can orded Cy5.5 labeled primer to do PCR to get your probe). Then you can use Odessy scanner to see it, without transfering  to membrane and even no need to take the gel out glass cassette.

On a second look of your gel, another scenario may be possible, would your probe only give out signal only after forming complex with protein? that is why there is no signal for free probe. In this case, is the unlabeled probe is your probe without the tag? it competes effectively there (no signal), whereas the mutant probe did not compete with your labeled probe (signal remains). How's that?