Institution: NIH Library Sign In as Member / Individual

This Article Abstract Full Text Services Alert me to new issues of the journal Request Copyright Permission

Supporting Information

Files in this Data Supplement:

Fig. 6. Calculated force (red curve) and corresponding spring constant (blue trace) for a worm-like chain polymer of persistence length 0.38 nm as a function of its normalized extension Z/L₀.

1. Modifying Antibody with N-Succinimidyl 3-(Acetylthio)propionate. (i) Wash PD-10 column with 30 ml of buffer A (100 mM NaCl/50 mMNaH₂PO₄/1 mM EDTA, pH 7.5). (ii) Load 200 m l (1 ml/ml) of antibody in 300 m l of buffer A onto PD-10 column. (iii) Pass 500 m l of buffer A through the column nine times. (iv) Collect fractions seven and eight and pool them. (v) Add a 10-fold molar excess of N-succinimidyl 3-(acetylthio)propionate (SATP)/DMSO to antibody solution for 30–60 min under argon. (vi) Rinse two PD-10 columns with 30 ml of buffer A. (vii) Add 500 ml of antibody solution into each PD-10 column, and then pass 9 ´ 500 m l of buffer A through the columns, collecting fractions seven and eight and storing them in 100-m l aliquots at –70° C.

2. Modifying Tips with APTES. (i) UV-clean MAC levers for 15 min. (ii) Purge a glass desiccator with argon for 2 min, and place 30 m l of aminopropyltriethoxysilane (APTES) (99%; Sigma–Aldrich) into it in a small container. Place 10 m l of N,N-diisopropylethylamine (99%, distilled; Sigma–Aldrich) into another small container in the desiccator and purge with argon for a further 2 min. Place cleaned probes into the desiccator, purge for another 3 min, and seal for 0.5 to 2 h. Remove reagents, purge with clean argon, and keep probes stored in this environment.

3. Crosslinker Reaction. (i) Mix crosslinker and 5 m l of triethylamine in 1 ml of CHCl₃. (ii) Place NH₂-modified tips into this solution for 2–3 h. (iii) Wash tips with CHCl₃, and dry with argon.

4. Linking SATP-Labeled Antibody to Tips. Incubate the tips in 50 m l of SATP-antibody, 25 m l of NH₂OH reagent (500 mM NH₂OH× HCl/25 mM EDTA, pH 7.5), and 50 m l of buffer A for 1 h. Wash tips with buffer A and PBS buffer (150 mM NaCl/5 mM Na₂HPO₄, pH 7.5) three times. Store tips in PBS buffer at 4° C.