Concentration of proteins in a crude preparations (such as culture sup) can be estimated semi-quantitatively by using "Dot Blot" method if you have both purified protein and specific antibody against it.
TBS: 20mM Tris-HCl, 150mM NaCl, pH 7.5
TBS-T: 0.05% Tween 20 in TBS
BSA/TBS-T: 0.1% BSA in TBS-T
Nitrocellulose membrane (BIO-RAD, Trans-Blot™ etc)
1. Have nitrocellulose membrane (NOT PVDF!) ready, draw grid by pencil to indicate the region you are
going to blot (see below).
2. Make serial dilutions of purified protein. For quantification of IgG, prepare the following solutions
0, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 ng/µl of mouse IgG in TBS
3. Make serial dilutions of your unknown sample, such as x1, x3, x10, and x30 dilutions for hybridoma
culture sup. Make control dilutions (fresh culture medium at the same dilutions) as well.
4. Using narrow-mouth pipet tip, spot 1µl of samples onto the nitrocellulose membrane at the center of the
grid. Minimize the area that the solution penetrate (usually 3-4mm diam.) by applying it little by little over
2-3 times of stroke of pipet.