Hi- wondering if someone out there can help figure out what's going on with my WBs!
So I have been transfecting one set of HuH-7 hepatocarcinoma cells with a plasmid of interest, and another set of HuH-7 cells with a negative control plasmid. These cells were lysed at 48 hours and at 72 hours.
I have been using Santa Cruz antibodies to seek out Protein S (72kDa), TFPI (40kDa), C4BP-a (70kDa) and C4BP-b (52kDa). Protein S is Mouse anti human, TFPI and C4BP-b are Goat anti human, and C4BP-a is Rabbit anti human. The proteins antibodies had to be optimised, and it was determined that TFPI is running higher than expected at about 50kDa. Actin was used as the housekeeping protein.
I have been using SDS-PAGE to transfer the proteins to a nitrocellulose membrane overnight (approx. 17 hours at 4 degrees Celsius). Then I checked each membrane with Poinceau Red staining, before applying a 3% Milk/TBS blotto for 90 minutes. The 3% is removed, and the Primary antibody (dissolved at the required concentration) in 1% Milk/TBST blotto is poured on for 90 minutes. The next step is to do 3x 5 minute washes of TBST, before pouring on the Secondary antibody/HRP and Streptavidin (again dissolved at the required concentration) in 1% Milk/TBST blotto. The Secondary antibody is left to wash the membrane for 90 minutes; it is then poured off, and the membranes undergo another set of 3x 5 minute washes. The membranes are blotted dry, and ECL fluid (from Bio-Rad) is pipetted onto them. The ECL fluid absorbs light for 5 minutes, and then the membranes are blotted again to remove the excess fluid before being sandwiched between two clean and clear plastic sheets. Finally, they get to be imaged at 30 second intervals for 2 and a half minutes.
During the optimisation process, the antibodies worked really well for Protein S, C4BP-a and TFPI. But then when I used the cell lysates, I end up with a non-specific band around 100kDa, across all antibody types. It's a very strong band. Protein S and C4BP-a both appear, however there are non-specific bands below them at about 60kDa. The TFPI and C4BP-b columns don't produce any bands.
The results are consistent across all the membranes I've trialled, however they aren't consistent with previous studies or with my optimisation blots. Sometimes the Molecular weight markers disappear entirely.
My supervisor and I are confused as to why a non-specific protein is binding to antibodies from 3 different species, and as to why the C4BP-b and TFPI don't show up, considering they are both linked to Protein S which is showing up very clearly.
Thanks in advance if you can think of anything, and if you seek clarification on anything, please ask!
(PS- I am still awaiting permission to upload the WB images)