Hey!
I have a problem concerning my Western Blots. Currently, I am working with a mutant protein that is really small (9 kDa protein) tagged with a FLAG-tag (1 kDa). I am using a semi-dry blotting system, 20% methanol, no SDS and a PVDF membrane with a pore size of 0.45 µm. Usually, I blot for 40 min at 280 mA. As I did not detect my protein, I changed the time to 15 min at 280 mA but after Ponceau staining there were still no bands visible (although the marker had a visible band at 10 kDa).
I already read some trouble-shooting guides, one suggesting that I put another PVDF-membrane behind the first one so even if there should be blotting-through, I could detect it on the second membrane. I also read that I could use a membrane with a pore size of 0.2 µm but as they are rather expensive I would start with something else.
I have no possibility to do a wet transfer and I would not put another tag on the protein to make it bigger.
I am very much looking forward to your input :)
Thank you!
Best,
Tatjana
Hi,
Have you tried a high percentage (such as 12% or as high as 20%) gel? That is better for such a small protein.
You can also stain the gel with coomassie blue to see if it's there prior to trying the transfer, and you can stain the membrane with ponceau red to see if protein is there.
Good luck!
Carmen
Hi,
I agree that using a thicker gel is the most efficient way of isolating smaller proteins. For the isolation of proteins ~10kDA, I would recommend using a 4% stacking gel, a 10% spacing gel, and a 16% separating gel, and run your samples on a large vertical electrophoresis unit (width × height, 16.5 × 14.5 cm) at 150V for 6 hours.
You were quite right in reading that a membrane of 0.2 µm would be best for blotting. I would recommend that you then transfer your proteins onto an Optitran reinforced nitrocellulose membrane (0.2 μm pore size) at 400 mA for 2 h at 4 °C.
The above protocol was developed in the below research:
http://www.ncbi.nlm.nih.gov/pubmed/20864542
Hope this helps!
Shane.
Tatjana, If the suggestions that you have already received do not work for you, depending on the composition of the protein you might want to try blotting in the presence of a cation. Several years ago we were having trouble blotting an 8kD protein and tried all of the suggestions that you have received to this point. A literature search turned up two papers that involved improved capture of small proteins in the presence of Ca++.
McKeon, T.A. and M.L. Lyman. 1991. Calcium ion improves electrophoretic transfer of calmodulin and other small proteins. Anal. Biochem. 193:125-130.
Mizzen, C.A., N.J. Cartel, W.H. Yu, P.E. Fraser, and D.R. McLachlan. 1996. Sensitive detection of metallothioneins-1,-2 and -3 in tissue homogenates by immunoblotting: a method for enhanced membrane transfer and retention. J. Biochem. Biophys. Methods 32:77-83.
Ca++ did not work for us, but we tried blotting in the presence of other cations and found that Zn++ worked best for us. It was like a miracle. It worked for our protein as well as for B-amyloid.
C Maillet, R K Gupta, M G Schell, R G Brewton, C L Murphy, J S Wall and B C Mullin Enhanced capture of small histidine-containing polypeptides on membranes in the presence of ZnCl2 BioTechniques 07/2001; 30(6):1224-6, 1228 - 1230. Let me know if you would like me to send the pdf of this paper.
Thank you all so very much for your input! I will try different things and hope for the best :)
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