They mentioned antibody and microbeads amount but there is no information about sample concentration in instruction manual. What should be my sample concentration??
2.2.1 Magnetic labeling
1. Add 1−2 μg of monoclonal antibody, 10−100 μL of hybridoma
supernatant (20−50 μg/mL), 0.1−1 μL of ascites (1−10 mg/mL),
0.5−5 μL of serum (containing 1−10% specifi c antibody), or 2−4 μg
of affi nity-purifi ed polyclonal antibody to the cell lysate. Mix well.
▲ Note: Pre-clearing the lysate with Protein A/G MicroBeads is usually not necessary.
2. • If monoclonal antibody, hybridoma supernatant, or ascites was
used for immunolabeling, add 50 μL of Protein G MicroBeads to
magnetically label the immune complex. Mix well.
• If serum or polyclonal antibody was used for immunolabeling, add
100 μL of Protein A/G MicroBeads to magnetically label the immune
complex. Mix well.
3. Incubate for 30 minutes on ice.
No! Step 1 says how much primary antibody we should use. I'm asking about sample concentration that includes target proteins. I'll use human serum as primary antibody. And my target is bacterial outer membrane proteins. So I have to calculate and adjust my sample concentration to prevent non-spesific interactions and masking by major proteins.
It depends on abundance of protein of interest in the sample and needs to be determined emperically. Use constant amount of antibody (1-2 ug) and add it to 10, 25, 50, and 100 ug of total protein in 1 mL reaction. Nutate the mixture overnight (12 h) at 4C. Now add the protein A/G beads and incubated on ice for 30 min.
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