I am performing experiment with C6 glioma cells which are known to express S100B usually according to ATCC data sheet. But when I perform western blot with the cell lysates, I fail to detect any bands at 10 kDa.
For lysis I used RIPA Buffer with PI mix.
For electrophoresis, I used 15% SDS-Page gel, Run: 100V, 60mA, 90mins. Transfer: 80V, 190mA, 60 mins.
I would like suggestions for this problem and also in detecting small molecular weight proteins.
Thank you.
How certain are you about the quality of the antibody? Do you include a positive control (eg a lysate prepared from cells over-expressed with protein of interest?
It can also be question of amount of lysate. You may need to load more than 50ug of total lysate to see poorly expressed proteins.
I am with Utkarsh. The methodology of the technique seems ok, maybe the problem is related with the antibody quality, protein amount or even membrane overblocking. Do you block the membrane with milk or BSA? Milk tends to hide low signals.
Dear Mohd
which membrane did you use?
PVDF? Nitrocellulose?
Which pore Size? 0,45, 0,22 or 0,10um?
It can be important when you work with small MW protein.
https://info.gbiosciences.com/blog/bid/203026/pvdf-or-nitrocellulose-which-membrane-is-best
Manuele
Dear Utkarsh Kapoor .
I have not checked with a positive control yet, and will keep that in mind.
Dear Juan Miguel Suárez-Rivero ,
I block with 5% Skim Milk. Have not tried with BSA yet.
Dear Manuele Martinelli
I use PVDF 0.45um wet transfer method.
Dear Mon e
i think that for a so small protein at least 0.22um is preferable. 0 45 has big pore and the protein can pass through the membrane.
ciao
Manuele
- For small proteins, I'd use tricine gels (doi:10.1016/0003-2697(87)90587-2), which resolve them better than standard SDS-PAGE.
- I'd also avoid detergent in the transfer buffer, which makes mobilisation of large proteins from a gel easier, but also may lead to small proteins slipping through the membrane.
- Methanol increases membrane binding of small proteins, but prevents mobilisation of large ones from the gel. You may also try the system from doi:10.1006/abio.1993.1313, which uses a methanol-containing buffer on the membrane side, but an SDS-containing on the gel side.
- Dunn's transfer buffer (doi:10.1016/0003-2697(86)90207-1, 10 mM NaHCO3, 3 mM Na2CO3) in my hands is better than Towbin's.
I used 0.2um PVDF and was able to detect my target protein of 10kDa. but the bands were not that clear, i mean smeared a bit. And did also load a positive control which came out but smeared. One thing I noticed was the time of PVDF membrane activation using methanol may be a factor which i am not sure about.
Any suggestions regarding membrane activation will be appreciated.
Haven't tried tricine gels yet so will have to try that too.
Thank you.
SDS can oligomerize and interfere with your signal at the bottom of the gel, where you protein is, impairing detection of a clean band. Have you considered ELISA?
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