Recently I keep getting similar issues with 4G10 platinum antibodies. When I try to look for total phosphorylation I keep getting these kinds of white spots on the blot. At first I thought these would be air bubbles during transfer, but the total protein on the blot looks fine, then I figured it has to be the incubation of either the primary or secondary, so I tried incubating with 10ml of antibody! in a bin, a tube and a bag. No difference, kept getting these white spots. I figured it could also be our CCD camera, but all the other images (same machine) look fine.
Anyone got any suggestions as to the cause of this?
Hello TIm. By white spot, are you referring to the void on the right side of the blot? To me that looks like a pocket of air or buffer between the gel and the membrane. We use a 12 x 75 mm tube like a roller to gently press the gel against the filter paper and then the membrane against the gel. The rolling motion displaces any trapped buffer or air without stretching or damaging either the gel or the membrane. I thin you effectively showed it was not due to uneven exposure to either primary or secondary antibody.
Hi Tim - Is this ECL detection? If so, it might be that you're membrane is drying out sometime during the ECL incubation or during imaging.
@Bruce There are no bubbles, the transfer is not the issue, the bottom image shows the membrane after transfer.
@Kyle Yes this is ECL, but during ECL incubation it in a plastic bag, and also after. I think it might actually be that the signal is too strong, since there is now also a precipitation on the membrane!
Too much signal can happen, but usually you see it as 'ghosting' of individual bands on the blot. Make sure no parts of the blot are stuck to the bag during incubation. I prefer to use these boxes (below) for ECL incubations, you can put it on a shaker to make sure nothing gets stuck/dried out.
https://www.hobbiesdepot.com/supplies/Plastic-Boxes
1. If you are using PVDF membrane, did you activated with methanol for 10" before the transfer? and after transfer if you dry your membrane: Rinse the membrane in water then dry 15 min at air, activate again by washing 10” in methanol and wash well in water before blocking. The rule is activate every time it dryes.
2. Use a good blocking buffer. If you use skim milk make sure it is fresh and filtered. I use starting block (Thermo Fisher) +Tween20. I got white regions one time I forgot to add Tween20.
3. Choose acrylamide % (or gradient) that allows you to see your protein of interest closer to the middle of the gel and well separated.
4. You are using p-tyr antibody. Well, that is dirty by definition since it is detecting every protein with a P-tyr in it. You might want to do an IP Western. You first IP your protein of interest and then detect with p-tyr. It will look very nice. Make sure you crosslink the antibody to the beads for excellent results.
@kyle, we have similar boxes, also had the same issue! The ghosting might be it, since I can now see the full protein pattern on my membrane after ECL incubation (precipitation of the ECL?)
@ soldedad, indeed it was PVDF and it was activated in methanol. It was never dried out during the procedure. I block with 5% BSA in TBS-T 0,45µm filtered. Separation is not the issue. We want to look for whole phosphorylation per sample on western blot, because we will do IP's but mass-spec it and not do WB on those samples.
- Badges
- Science method