I want to detect AIM2 (39kDa) expression in response to treatment in a cell line. I got something like the attached photo consistently in several repetitive experiments. The ECL signal intensity is much higher on the right that it even stains the ladders but not the ones on the left. It happened to my beta-actin (43 kDa) loading control as well but, curiously, when I switch to a smaller loading control, profilin (15 kDa), this effect is almost completely gone.
Initially I suspected that it was the transfer that went wrong. I tried semi-dry and dry transfer but still got the same problem. Does anyone know what was going wrong?
Dilutions:
AIM2 1:500
Beta actin: 1:2000, 1:5000, 1:10,000 still the same
profilin 1:2000
all secondary 1:2000
Hi,
I still think it could be a transfer issue, if it's MW-dependent. To check this, you can stain the blots with Ponceau S or Amido black after transfer. In case of Amido black, you can stain the blots even after the detection.
Another possibility will be the optics; either illumination, filters, or the camera. You can test this by moving or rotating the blots on the stage.
The third possibility will be the incubation with antibodies. Although it does not seem likely from your photos, it will be worth checking if the liquids move well during the incubation.
Hi,
I still think it could be a transfer issue, if it's MW-dependent. To check this, you can stain the blots with Ponceau S or Amido black after transfer. In case of Amido black, you can stain the blots even after the detection.
Another possibility will be the optics; either illumination, filters, or the camera. You can test this by moving or rotating the blots on the stage.
The third possibility will be the incubation with antibodies. Although it does not seem likely from your photos, it will be worth checking if the liquids move well during the incubation.
It does look like a transfer problem and could be equipment related. can you use capillary transfer the old fashioned ( but well tested) route ?
Thanks all,
Re Shin-Ichiro, I have tried rotating the blots but yielded the same results. I also cling-wrapped every incubation tray and made sure they sit properly on the oscillator. So I guess I should stain the blot after transfer to see if I got problems with the transfer.
Re Paul, the two transfer methods have been well optimized by my colleagues and they have produced many good results for us, including myself! Even those who routinely do western blotting in my lab have no idea why there is a problem all of a sudden. If this issue is equipment related then how would it be? Unfortunately no one in my lab has done the old method so not sure about that.
I agree that this is most probably a transfer issue. As you will know, smaller proteins are more easily eluted from the gel and transferred to the membrane, shorter transfer time or lower current is needed for small proteins. So even if you have reduced transfer efficiency at one end of the gel, it might still be sufficient for complete transfer of small proteins. You mentioned that the blotting method is well established in your lab, so it should not be a general problem of the method. Is the equipment you have used also used by other people in the lab at the moment? Are they experiencing similar problems does it perform good in their hands? There might be a loose contact or some kind of deterioration of the electrode. This could be tested by changing the blotter if you have a second one. If other lab members dont experience similar problems, you could ask another person to do the experiment once and compare the outcome, to find out if there is some small mistake in the way you do it which you might not think of.
is it possible that the problem occurs earlier at the electrophoresis stage. If the gel casting is distorted and in the faint lanes the sample has run down the outside of the gel not through the gel so has ended up in the buffer reservoir. Is this a clamping issue perhaps. testable if one of your colleagues will pour a gel for you.
Thanks again,
Re Wolfgang, when I was using the Xcell blot module transfer, I tried both possible orientations (long edge to the bottom or long edge to the side, as in rotating the sandwich 90 degrees) but they yielded the same results. Does that mean I did not have uneven transfer efficiency across the blot?
2 of my colleagues are doing western blot recently but only one of them had similar problems. The three of us used the same equipment (so it should not be the blotter) and we even did one of the blots together but no one spotted any technical issues that would cause the transfer problem.
Re Paul, thanks for replying again. We used commercial gels so presumably there shouldn't be a problem with gel being distorted. I even tried a different batch of gels of the same company to see if it was a batch-dependent issued but it wasn't. These gels worked well for me and everyone else in the past so it would be horrifying to think that they are problematic all along!
if rotating the gel in the blotter gives the same result I still feel that the problem can be in the electrophoretic separation.something like the gel temperature rising and some leakage happening as the sample runs into the gel.WHen you take the gel off the e/f rig ,or even while running, do the intensities of the loading dye bands look the same intensity or do they mimic the transfer intensities?.If the latter then you have some form of leak in the system I think.
Hello Aden Ho Yin Lau, I have exactly the same problem as you!
I measure the protein conc. with BCA assay and load the same amount of protein in each well. But it looks like there is higher signal intensity detected with the sample that is placed on the right side of membrane.
When I load the same sample to left side of the well, it seems like it showed not "higher" but right signal intensity. Meaning the signal intensity differs depending on the well.
I wonder if you solved the problem?
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