HRP tag.

asked:
have any friends who make spicy root peroxide mark ah, can you send me your steps. I began to do, found that many people use different concentrations.
:
1.1
Reagents
Preparing
1) 10mg/mL HRP
2) 0.06mol/L NaIO4
3 0.16mol/L glycol
4) carbonate buffer (Na2CO3 0.7952g NaHCO3 1.4702g dissolved in 500mL Distilled water, adjust pH to 9.5)
5) 2.5mg/mL NaBH4
6) 0.01mol/L phosphate buffer, pH7.4
1.2 Operation step
1. 10mg/mL to take HLP VmL, the solution is brownish yellow, add 0.06mol/L NaIO4 VmL, immediately turn gray-green. Set aside 30min, 4C
2. Add 0.16mol/L glycol VmL, the solution changes back to brownish yellow, RT, set aside 30min.
3. Add
antibodies
Vml 5mg, CB, 4C dialysis overnight.
4. Add 5mg/mL NaBH4 VmL and the solution is bubbled. Leave 2h at room temperature.
5. 50% saturated ammonium sulfate salt analysis once, 12000 rpm, 4 degrees C centrifugation 30min. Precipitation is reseated with 5VmL 0.01mol/L PB. Load the
in a dialysis
.
6. The solution was dialysis overnight at 0.01mol/L PB, pH7.4.
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