Hello,
I was looking for some advice on an issue I am experiencing with some recent Western Blots. I am looking at two proteins, one ~10 kD and the other ~ 5 kD.
Conditions:
- 16.5% pre-cast Tris/Tricine gels
- semi-dry transfer at 15V for 20 min with buffer containing 192 mM glycine, 25 mM Tris, and 20% methanol, have been using 0.45 uM PVDF membrane (we just got some 0.2 uM so I will try that as well due to small protein size), activated with methanol
- blocking with 3% BSA (1 hr room temp), primary and secondary antibodies diluted in 3% BSA as well (overnight 4C for primary, 1 hr room temp secondary)
- LiCor Odyssey imaging system
I have been optimizing the transfer conditions and it seems like they are finally working. I can visualize the bands on the membrane after transfer with Ponceau stain (see photo). However, when I proceed with Western Blot, the bands end up grainy/fuzzy, and for the 5 kD protein, you can't really see it at all despite being able to see the bands with the less sensitive Ponceau stain (see photo). In the photo, I have the settings brightened a lot to see anything so there is more background visible, and the red at the bottom is just the dye front from the gel. The antibody for the 5 kD protein is prepared at 1:500 and the antibody for 10 kD protein at 1:1000. This gel has 3, 6, 12, 24, or 40 ug of the 5 kD protein on the left, and 0.4, 0.8, 1.6, 5, or 20 ug of the 10 kD protein on the right.
I have previously gotten much better blots for the 10 kD protein using the same antibody/same dilution, etc, and was able to see bands at low concentrations including the ones used here and even less. The smaller protein has been more problematic but I got it to work at least once before and I was more optimistic this time around given the Ponceau stain result. That the 10 kD protein is not working well in Western Blot now is making me think there is something else going on.
Any thoughts or suggestions on the reasons for this type of band upon Western Blot despite better Ponceau stain result?
Thanks!
As shown in image and your details, it seems that the despite having 1:500 dilution the primary antibody did not bind to 5 kD protein of your interest. It seems that the issue may be with the primary antibody of protein of your interest. I would suggest to use a fresh antibody for 5 kD protein with the same dilution to rule out this possibility.
It seems to me that either your primary antibody for the 5 kDa protein is completely dead, or that you may have a problem due to fluorescence quenching by the Ponceau red dye. This would make sense since Ponceau red absorbs with a maximum around 520 nm, i.e. probably close to the emission wavelength of your green fluorescing antibody.
Thank you Shail and Pierre for your suggestions. I have been preparing the primary antibody fresh for each assay, so hopefully this would prevent an issue with the antibody. I have used it successfully before at this dilution, but maybe it is possible something has happened to the Ab over time. The 10 kD protein antibody is also prepared fresh and has worked for very low concentrations of the protein previously, so the fact that is not working for several of these bands is odd. We will try with skipping the Ponceau staining now that we saw those transfer conditions are working, in case there is fluorescence quenching or something else going on to due the staining. Thanks for the advice!
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