•Samples (1, 2, 3, 4 in the figure) are mouse brain tissues.
•Primary Ab is mouse IgG.
•Secondary Ab is anti-mouse IgG-HRP.
•Correct brand size (91kb) is detected, but there are non-specific brands which are the almost same size in Secondary Ab only treated membrane.
I think the Secondary Ab non-specifically binds to samples because the samples are mouse tissue.
I follow the procedure below
•Blocking in 6% skim milk (in TBS-T) for 30 min rocking (21rpm) at 4℃
•Incubation with Primary Ab diluted in 6% skim milk overnight rocking (21rpm) at 4℃
•Washing three times in TBS-T for 10 min each washing rocking (21rpm) at 4℃
•Incubation with Secondary Ab diluted in 6% skim milk for 1hr rocking (21rpm) at 4℃
•Washing three times in TBS-T for 10 min each washing rocking (21rpm) at 4℃
•ECL reaction and imaging
hi
you can Pre-adsorbed tissue lysate before loading by sepharose or agarose-protein A/G bead in this test ( mouse tissue IgG captured by protein A/G). for this purpose you should prepared lysate in "Non-denaturing lysis buffer"
"Non-denaturing lysis buffer"
Use for antigens that are detergent soluble and are recognized in native form by the antibody. Triton X-100 can be substituted for NP-40.
- 20 mM Tris HCl pH 8
- 137 mM NaCl
- 1% Nonidet P-40 (NP-40)
- 2 mM EDTA
Store up to 6 months at 4°C. Immediately before use add protease inhibitors.
For convenience, a 10% sodium deoxycholate stock solution (5 g into 50 mL) may be prepared. It must be protected from light.
good luck
@Reza Hadavi Thank you for your supportive advice:) I will get the information about agarose A/G bead more.
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