Hi. I'm trying to extract IFN-G from transfected CHO cells. I have performed western blots a few times with both transfected and non-transfected CHO cell lysate samples, and I'm not getting consistent results. Here are some issues which I'm facing.
1. multiple bands are seen. some bands are also observed for non-transfected cells.
2. same concentration of antibodies are not giving the similar scan images when repeated.
3. sometimes random black spots are observed on the membrane.
The gene expression of both transfected and non-transfected cells is confirmed using q-PCR.
Can anyone provide any suggestion or solution?
Reprobing Westerns is fine. To make sure all primary AB is gone after stripping, probe with secondary AB only. Unless there was an error, statistics won't improve, though, rather the opposite, as background noise will increase.
Multiple bands are seen frequently. Alternative splicing, cross-reactivity or post translational modification might be the culprits.
Quantitative Westerns are tricky, unless you have access to a ECL or fluorescence scanner that has a linear sensitivity over several (5 or 6) orders of magnitude.
The spots you see probably are dust, human keratinocytes, or particles from powdered gloves.
Are you aware that IFN-G is secreted into media? Maybe better opt for an ELISA.
As Wolfgang said, IFN-g is released rather than expressed on a cellular surface; therefore, this could be the reason why you don't get consistent results (ELISA would be a better option to quantify IFN-g). The qPCR results will likely be more consistent due to the fact that you are dealing with intracellular material rather than secreted cytokines.
1. If you get extra bands, as Wolfgang mentioned, it could be PTMs or cross-reactivity; if the latter is true, maybe you could try changing the primary antibody? Try to use a monoclonal antibody, or an antibody tested in the literature which shows nice results.
2. This might sound obvious, but are you making sure you use the same scanning parameters each time? Also, if you re-use a primary antibody many times, at some point I would assume that it will lead to a reduction in band intensity (because the more you use it, the less antibody will be left in your solution).
3. Maybe the spots could be clumps of milk in the antibody solution (if you use milk?); I had this problem and since I started filtering the milk/TBS-T prior to use, it never happened again.
Thank you so much for your suggestions. I could try filtering the milk next time. And yes, we have PVDF as well. I'll using that for my next experiment.
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