Goat anti-mouse IgG (100 µL, Jackson ImmunoResearch, product number 115-005-146 (Fig. 4c ) or Thermo Fisher Scientific, product number 31160 (Fig. 6a and Supplementary Fig. 12 )) was dialyzed against conjugation buffer (150 mM NaCl and 10 mM HEPES, pH 8) at room temperature and 4 °C (3 × 300 mL; 2 hours, 2 hours, and overnight), using a 10-kDa molecular weight cut-off dialysis cup (Thermo Scientific). A ~ 1 mM solution of Eu3+/DTBTA or Alexa Fluor 594 NHS ester (Thermo Fisher Scientific, product number A20004) in ice-cold conjugation buffer was prepared immediately before use, and 5 µL of this solution was added to 100 µL of the dialyzed antibody solution. After the reaction mixture was incubated at room temperature overnight, unreacted Eu3+/DTBTA was removed by two rounds of size-exclusion chromatography (Illustra MicroSpin G-50; GE Healthcare Life Sciences). The resulting stock solution of Eu3+/DTBTA-labeled and Alexa Fluor 594-labeled secondary antibody appeared red-fluorescent upon 365-nm illumination. The probe-to-antibody ratios were calculated from absorbance levels at 341 nm (Eu3+/DTBTA), 594 nm (Alexa Fluor 594), and 280 nm (antibody) as determined with a NanoDrop spectrophotometer (Thermo Scientific).
Illustra microspin g 50
Manufactured by GE Healthcare
The Illustra MicroSpin G-50 is a DNA purification column designed for the rapid and efficient purification of PCR and other DNA fragments from enzymatic reactions. It utilizes a size-exclusion chromatography method to remove unwanted salts, nucleotides, and other low molecular weight contaminants from DNA samples.
Automatically generated - may contain errors
4 protocols using illustra microspin g 50
1
Fluorescent Antibody Conjugation Protocol
2
Eu3+/DTBTA Labeling of Dextran Amine
Check if the same lab product or an alternative is used in the 5 most similar protocols
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10-kDa dextran amine (1 nmol; Molecular Probes) in 7.5 µL conjugation buffer (150 mM NaCl and 10 mM HEPES, pH 8) was added to a 1.2 mM solution of Eu3+/DTBTA (7.5 µL) in the same buffer. The reaction mixture was incubated at room temperature for 3 h in the dark. Excess Eu3+/DTBTA was then removed by size-exclusion chromatography (Illustra MicroSpin G-50; GE Healthcare Life Sciences).
3
Fluorescent Antibody Conjugation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Goat anti-mouse IgG (100 µL, Jackson ImmunoResearch, product number 115-005-146 (Fig. 4c ) or Thermo Fisher Scientific, product number 31160 (Fig. 6a and Supplementary Fig. 12 )) was dialyzed against conjugation buffer (150 mM NaCl and 10 mM HEPES, pH 8) at room temperature and 4 °C (3 × 300 mL; 2 hours, 2 hours, and overnight), using a 10-kDa molecular weight cut-off dialysis cup (Thermo Scientific). A ~ 1 mM solution of Eu3+/DTBTA or Alexa Fluor 594 NHS ester (Thermo Fisher Scientific, product number A20004) in ice-cold conjugation buffer was prepared immediately before use, and 5 µL of this solution was added to 100 µL of the dialyzed antibody solution. After the reaction mixture was incubated at room temperature overnight, unreacted Eu3+/DTBTA was removed by two rounds of size-exclusion chromatography (Illustra MicroSpin G-50; GE Healthcare Life Sciences). The resulting stock solution of Eu3+/DTBTA-labeled and Alexa Fluor 594-labeled secondary antibody appeared red-fluorescent upon 365-nm illumination. The probe-to-antibody ratios were calculated from absorbance levels at 341 nm (Eu3+/DTBTA), 594 nm (Alexa Fluor 594), and 280 nm (antibody) as determined with a NanoDrop spectrophotometer (Thermo Scientific).
4
Eu3+/DTBTA Labeling of Dextran Amine
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