Poly-HRP Streptavidin Conjugate-Femtotracer recommendations to use
Chapter 1. Buffer systems
(1) Use 150-200 mM NaCl ionic strength in all assay buffers.
(2) Preferably Tris based assay buffer 25-100mM pH 7,8-8,2.
(3) Use 0,1-0,5% casein as a protein additive in all assay buffers, try different suppliers and type of casein.
Recommended Casein:
Sigma #C3400 / MP#901293
the protein should be converted to the sodium salt by stirring 50 g/L suspension overnight or longer with 0,3 M NaOH, neutralize by HCL to pH8-8,5 prior to use, store at +4C with 0,1% Proclin300 as a preservative
(4) Avoid casein hydrolysates like tripton or peptone.
(5) Include 0,05-0,25% nonionic detergents.
Recommended detergent:
0,05-0,25% Triton X100 or X705.
(6) Look for other detergents additives, compatible to your assay, detergents have a strong effect and can be very helpful.
(7) Use plate blocking agents based on casein, try different sources.
Recommended blocking solution for the plates:
0,1% Casein (Sigma C3400)/1% Sucrose. HRP stabilizer from SURMODICS (USA)
(8) Use 2 mM CaCl2 as a peroxidase stabilizing additive at all assay buffer.
(9) Do not use EDTA in the assay buffer, avoid any other chelating agents or buffer components that can make a complex with Ca ions, it will affect the HRP.
(10) Do not use sodium aside in the assay buffers, it will destroy the HRP.
(11) Take care about traces of free biotin in your buffer composition, it will interfere with your assay.
(12) Use proven peroxidase-safe preservatives like ProClin 300/900 1:500....1:1000 dilution factor.
Chapter 2. Working dilution
(1) Typical working (assay) concentration for PolyHRP-SA is 1-0,25 mkg/ml.
(2) Titrate your biotinylated detection Ab or Ag together with PolyHRP-SA, use precise chessboard titration mode to find optimal concentrations combination of both reagents.
(3) Use shaker incubations if possible.
(4) Recommended incubation time with shaker is 10-15 min and 20-30 min without shaking.
Chapter 3. Other notes of use
(1) Avoid opened sources of hydrogen peroxide or other chemically active vaporing agents in your lab - it can affect PolyHRP-SA and cause high assay background.
Chapter 4. Stability
(1)Typically, stability drops down on 20-30% during first 10-12 months of real time storage at +4C depending of stabilizer used.
Recommended commercial stabilizers :
Original PolyHRP stabilizers from polyHRP.com
RP stabilizer from SURMODICS (USA) www.surmodics.com
(2) Take in to account 10-15% activity drop soon after the first dilution from the glycerol stock.
(3) PolyHRP-SA product can be lyophilized at any concentration. We have specially developed stabilizer for lyophilisation, please order on PolyHRP.com.