Antigen Information
- P15976
- 2623
- ERYF1
- GATA1
- GF1
- Human
Assay Format
- Human
- Cell Lysates
- Nuclear Extracts
- Sandwich-based
- Semi-Quantitative
Product Specifications
Introduction
Product Features
- Specific transcription factor-DNA binding assay
- Perfect alternative to EMSA
- Easy to perform in an ELISA format
- Non-radioactive assay
- High throughput (96-well plate format)
- Assay can be completed within 5 hours
Application Notes
- 96-well Strip Microplate pre-coated with DNA probes
- DNA Binding Buffer
- Positive Control Sample
- Specific Competitor DNA probe
- Non-specific Competitor DNA probe
- Assay Reagent
- DTT
- Wash Buffer
- Primary Antibody
- HRP-conjugated Secondary Antibody
- Antibody Diluent Buffer
- TMB One-Step Substrate Reagent
- Stop Solution
- Distilled or deionized water
- 100 ml and 1 liter graduated cylinders
- Tubes to prepare sample dilutions
- Absorbent paper
- Precision pipettes to deliver 2 µl to 1 ml volumes
- Adjustable 1-25 ml pipettes for reagent preparation < li="">
- Microplate reader capable of measuring absorbance at 450 nm
- Prepare all reagents and samples as instructed in the manual.
- Add 100 µl of sample or positive control to each well.
- Incubate 2 h at RT or O/N at 4 °C.
- Add 100 µl of prepared primary antibody to each well.
- Incubate 1 h at RT.
- Add 100 µl of prepared HRP-secondary antibody to each well.
- Incubate 1 h at RT.
- Add 100 µl of TMB One-Step Substrate Reagent to each well.
- Incubate 30 min at RT.
- Add 50 µl of Stop Solution to each well.
- Read at 450 nm immediately.
Typical Data
Figure 1Transcription factor activity assay of GATA-1 from nuclear extracts of K562 cells or HeLa cells. In K562 cells, activated GATA-1 is translocated into the nucleus where it binds with its corresponding DNA. A. Western-blot result of GATA-1 from cytoplasmic and nuclear fractions. B. Transcription factor activity assay of GATA-1 from nuclear fractions with the RayBio® GATA-1 Transcription Factor-Activity Assay Kit.
Figure 2Transcription factor activity assay of GATA-1 from nuclear extracts of K562 cells or HeLa cells with the specific competitor or non-specific competitor. The result shows specific binding of GATA-1 to the GATA conserved binding site detected by using the RayBio® GATA-1 Transcription Factor-Activity Assay Kit.
Storage/Stability
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多谢!
不知道能不能直接买到大鼠抗豚鼠血管IgG的ELISA试剂盒。
如果买不到这种试剂盒,用空白板的话(就是反应孔中没有抗原或者抗体包被),把自制的豚鼠血管抗原加到反应孔中进行孵育,能不能使抗原附着在孔壁上?这样的话就可以按照elisa的操作步骤进行大鼠血清检测了。
目前的设想是自制抗原,粉碎豚鼠的血管,制成悬液,通过反复多次的冻融,离心后收集上清液作为抗原(即豚鼠血管抗原)。
然后将抗原加入到elisa的反应孔中(这种是特制的空白板,就是反应孔中没有抗原或抗体包被)进行孵育,使豚鼠血管抗原附着在孔壁上,就是让反应孔充当固相载体,形成固相抗原。倒掉多余的抗原。
再加入待检测的大鼠血清,这样血清中的特异性抗豚鼠血管IgG就可以跟固相的抗原结合,形成固相抗原抗体复合物。
加入酶标的兔抗鼠或者羊抗鼠IgG,形成酶标的抗原抗体复合物。
然后就是一些显色步骤。
现在关键的问题就是抗原加入空白板,能不能形成固相抗原,如果不能跟孔壁附着的话,在后面洗涤的过程中就被洗掉了,那就没办法完成检测了。
请各位大侠给指条明路啊!!!小弟在此多谢了!!!
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