Human LBP PicoKine™ ELISA Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Store at 4°C for 6 months, at -20°C for 12 months. -Avoid multiple freeze-thaw cycles. -The standard solutions are best used within 2 hours.	-The diluted Avidin-Biotin-Peroxidase Complex (ABC) solution should not be prepared more than 1 hour prior to the experiment. -Prepare the substrate solution immediately before use or bring the pre-made substrate to room temperature. -Sodium azide is an inhibitor of HRP. Do not include the azide in buffers or wash solutions if HRP-labeled conjugate is used for detection.

Upstream tips
-Store at 4°C for 6 months, at -20°C for 12 months. -Avoid multiple freeze-thaw cycles. -The standard solutions are best used within 2 hours.
Protocol tips
-The diluted Avidin-Biotin-Peroxidase Complex (ABC) solution should not be prepared more than 1 hour prior to the experiment. -Prepare the substrate solution immediately before use or bring the pre-made substrate to room temperature. -Sodium azide is an inhibitor of HRP. Do not include the azide in buffers or wash solutions if HRP-labeled conjugate is used for detection.

Publication protocol

The effects of peptides on the interaction between LPS and low concentrations of LBP were determined as previously described20. Briefly, the plates of a LBP ELISA kit (Boster EK1274) were blocked by the incubation of 1% BSA prepared in 10 mM sodium phosphate buffer (pH 7.4) at 37 °C for 1 h, which were subsequently washed with 0.1% Tween-20. Aliquots of 100 μL of mouse LBP (Sigma SRP6034, 60 ng/mL) were added and incubated at 37 °C for 1.5 h. Peptides were prepared in sterile water, with concentrations of 12.5, 25, and 50 μg/mL, respectively. Biotinylated LPS (50 ng/mL) was added in the absence or presence of peptides (pre-incubated at 37 °C for 30 min). Polymyxin B (200 ng/mL) was used as a positive control. The binding of LPS to LBP was determined using a chromogenic reaction between HRP-conjugated streptavidin (Boster BA1088) and TMB (Sigma 860336). Data were processed by subtracting the absorbance in the absence of LPS. Statistical significance was determined by the Student−Newman−Keuls multiple comparisons test. The experiment was conducted in duplicate and repeated three times.

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Manufacturer protocol

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