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Inquiry: IGF-1 is usually bound to IGFBP, is it necessary to do an extraction before doing ELISA? I have read that IGFBP usually interferes with IGF-1 assay. Is there a step in removing the BP's How efficient is the kit in removing the IGFBP
Asked on Aug 27 2014
This kit is designed to measure free IGF1. As you stated, IGF-1 is usually bound to IGFBPs in the serum/plasma, so if you would like to measure total IGF1, it is advisable to release the analytes by performing an extraction procedure such as acid-ethanol treatment (Please see below for recommended sample pretreatments).
Reagents to Pretreat Serum/Plasma Samples
Acid-ethanol extraction solution: add 2.5 ml hydrochloric acid (37% HCl) into 10 ml deionized or distilled water, and then add ethanol to bring up to final total volume 100 ml. Mix well.
2 M Tris buffer (pH=7.6): Dissolve 24.2 g Trizma base in 70 ml deionized or distilled water. Adjust to pH=7.6. Bring up to final total 100 ml.
Sample Pretreatment Procedure
To release IGF-I from binding proteins, please follow the procedure outlined below. The following procedure is only for serum and plasma sample pretreatment. Do not pretreat cell culture supernatant samples or standard.
1. Add 30 μl serum or plasma to 120 ul acid-thanol extraction solution in a polypropylene tube.
2. Vortex and incubate for 30 min at RT with shaking.
3. Centrifuge 5 min at 10,000 rpm and transfer 100 ul supernatant into 200 ul Tris buffer (pH=7.6) in a polypropylene tube. Mix tube thoroughly.
4. Add 300 ul Assay Diluent A. Mix well. Assay immediately.
Note: Since the samples are pretreated, the concentration read from the standard curve must be multiplied by the dilution factor, 30.
Sam WasherAbcam Scientific Support
Answered on Aug 27 2014