The thing is that I am transfecting both protein isoforms separately in 10 cm dishes with COS-1 cells and FuGENE® HD Transfection Reagent. I am transfecting 3 µg of the each vector containing each isoform with 12 µl of the FuGENE® HD Transfection Reagent (1:4 ratio). I am lysing the cells with the NP-40 lysis buffer. I'm using a first mouse anti-Myc tag antibody and a second peroxidase labelled anti-mouse antibody. Well, the point is that at the end I get a strong band signal for the bigger 36.7 kDa protein isoform but I don't get any signal at all for the smaller 15.6 kDa protein isoform. I cannot detect this last one. I sequenced both vectors and both myc tags are perfectly in frame. I also performed a Beta-Tubulin Loading Control and that's not definitely the problem. The Cell line used should be the optimal one.
P.D. I am talking about a cytoplasmatic (soluble) protein. Any suggestions?
The power conditions depends heavily on composition of transfer buffer. For example, the more SDS in buffer added leads to the shorter transferring time. I use 250 V and max current (~400 mA) for 40 min for transferring 9,5 kDa protein onto 0.22 um PVDF membrane. For optimization of transferring time it is useful to use Prestained Protein Ladder (eg https://www.thermofisher.com/order/catalog/product/26616) for control of transfer process, like we in our lab do. And you may put the second one PVDF membrane with the first one: if the transfer time is too long, the protein will stop onto the second membrane.
Hi Ivan,
The condition that I use for Western Blot is 30 V, 3 A, 300 W for 1.5 hour. The size of my protein is around 40 kDa.
Best,
Najad
The power conditions depends heavily on composition of transfer buffer. For example, the more SDS in buffer added leads to the shorter transferring time. I use 250 V and max current (~400 mA) for 40 min for transferring 9,5 kDa protein onto 0.22 um PVDF membrane. For optimization of transferring time it is useful to use Prestained Protein Ladder (eg https://www.thermofisher.com/order/catalog/product/26616) for control of transfer process, like we in our lab do. And you may put the second one PVDF membrane with the first one: if the transfer time is too long, the protein will stop onto the second membrane.
Thanks Syed A Ali,
For the time being we don't have 0.2 um PVDF membrane. I just have a 0.45 micron PVDF membrane. Since my protein of interest is 15.6 kDa weight, Perhaps I could reduce the time and current parameters during the Trasnfering in order to not to lose my protein. Or even put 2 membranes to not let it pass through if that is the real problem. With a 0.45 micron PVDF membrane and a 15.6 kDa protein which time and current parameters would You suggest me for the transfering step?
Thanks
Hello Ivan,
Transfer parameters vary considerably from instrument to instrument and transfer conditions (wet, semi-dry, and dry) and must be determined emperically. As suggested by Ivan Bogdanov and yourself, using two membranes can do the trick. Although less popular and almost unknown to new gen researchers, simple diffusion (diffusion blotting) can also be used to transfer proteins. Since no electric current is involved, transfer is not as efficient (~10% compared to electro blotting) but if enough protein is present it does work pretty well. However, diffusion -based transfer is only qualitative and not quantitative. I have never worked with such low molecular weight proteins but my hunch is, diffusion can be used to transfer your protein without fear of it getting passed through the membrane.
Are you making sure you are using a high percentage (12-15% acrylamide) gel to resolve those bottom bands and also not letting anything run off? We include SDS only in running buffer, not in transfer. 0.2um PVDF is probably what you need, although I found this on another message thread which suggests a short 60min transfer:
"Hi,
I frequently use WB to detect and quantify proteins that are 10-15kDa with a PVDF 0.45um membrane (Immobilon) and I never had a problem. I normally transfer at 400mAMP for 60 minutes. With low molecular weight proteins, you obviously need to ensure that your transfer is no longer than 1 hour (at 400mAmp). ..., this also depends on how much proteins you have loaded, how abundant is your protein and also how specific and sensitive is your detection antibody. But as far as the transfer is concerned, PVDF at 0.45um is not a problem with 10-15kDa proteins.
Good luck."
did you probed any other protein coming to 15kd range, if that is not coming may be your transfer or gel percentage problem. if you getting 15kd range protein, then the problem may be your myc antibody is not detecting the protein. You can try another myc antibody.
Good luck
Our lab regularly ttanfers an 11kDa protein. No SDS in our tranfer buffer, just tris, glycine. & 20% MeOH. We use 0.2um nitrocellulose, but if you need to go with what is on hand, use 2 membranes, just in case. I've had success tranfering for 1 hour @ 100V, as well as at 20V for 18 hours. We useca constant voltage setting.
I am studying exogenously expressed protein fragments of 13.5-19 kDa and an endogenous protein of 34 kDa. I run in 15% SDS-PAGE with 1.5 mm thickness; transfer to 0.2 um PVDF membrane in regular transfer buffer but with 10% methanol. Transfer condition is 110 V for 50 minutes.
I have tried 110 V for 30, 40, 45, 50, and 60 min. The best is 50 minutes.
Besides, I also tried one and two 0.45-um PVDF membrane with different transferring time. However, it was very difficult to probe proteins with
As said in previous suggestions the problem may not only rely in the transfer but also in the elecrtophoresis process. Such a small protein (~15 kDa) may be running off the gel where the control (~50 kDa) is retained. For a 12 kDa protein we used a 10-20% gradient gel. If you are already sure your 15 kDa protein is in the gel, a 40 min wet transfer at 100 V without SDS will work for transferring both isoforms. Anyway I hope by this time you've already solved that issue.
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