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      第一章 細胞因子及其ELIS
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      附錄一 常用實驗方法
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      染色質免疫沉淀(X-ChIP)
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      染色質免疫沉淀(X-ChIP

      1. Cross-linking and cell harvesting

      1.1. Start with two confluent 150 cm2 dishes (1×107- 5×107 cells per dish). Cross-link proteins to DNA by adding formaldehyde drop-wise directly to the media for a final concentration of 0.75% and rotate gently at room temperature (RT) for 10 min.*

      1.2. Add glycine to a final concentration of 125 mM to the media and incubate with shaking for 5 min at RT.

      1.3. Rinse cells 2 x with 10 ml cold PBS.

      1.4. Scrape cells into 5 ml cold PBS and transfer into 50 ml tube.

      1.5. Add 3ml PBS to dishes and transfer the remainder of the cells to the 50 ml tube.

      1.6. Centrifuge for 5 min at 1,000 g.

      1.7. Carefully aspirate off supernatant and resuspend pellet in FA Lysis Buffer (750 μl per 1×107 cells).

      * When using suspension cells, start with 1×107- 5×107 cells and treat with both 0.75% formaldehyde

      and glycine as described above. Pellet cells by centrifugation (5 mins at 1,000 g). Wash 3 x with

      cold PBS and resuspend pellet in FA Lysis Buffer (750 μl per 1×107 cells). Proceed to Step 2.1.

      2. Sonication

      2.1. Sonicate lysate to shear DNA to an average fragment size of 500 to 1000 bp. This will need optimizing as different cell lines require different sonication times. Follow the fragment size on a 1.5% agarose gel.

      2.2. Centrifuge for 30 secs, 4°C, 8,000 g and transfer supernatant to new tube.**

      2.3. Remove 50 μl of each sonicated sample This sample is the INPUT, and this is used for obtaining the DNA concentration (see Step 5).

      **The lysed cells can be snap frozen in liquid nitrogen after and stored at -70°C for up to 2 months.Avoid multiple freeze-thawing.

      3. Immunoprecipitation

      3.1. Use approximately 25 μg of protein per IP. Protein concentration can be calculated using the Bradford assay. Dilute each sample 1:10 with RIPA Buffer. You will need one sample for the specific antibody, and one sample for the beads only control.

      3.2. Add the primary antibody to all samples except the beads-only control. The amount of antibody to be added has to be determined empirically, 1-10 μg of antibody per 25 μg of protein often works well.

      3.3. Add 20 μl of protein A/G beads (pre-absorbed with sonicated single stranded herring sperm DNA at 1.5 μg / 20 μl beads***) to all samples and IP overnight with rotation at 4°C.

      3.4. Centrifuge the protein A/G beads for 1min at 2,000g and remove the supernatant.

      3.5. Wash beads 3 x with 1ml Wash Buffer (centrifuge as above).

      3.6. Wash beads 1 x with 1ml Final Wash Buffer (centrifuge as above).

      ***Preparation of protein A/G beads with single stranded herring sperm DNA

      - Mix an equal volume of Protein A and Protein G beads and wash 3 X in RIPA Buffer.

      - Aspirate RIPA Buffer and add single stranded herring sperm DNA to 75 ng/μl beads and BSA to a final concentration of 0.1 μg/μl beads. Add RIPA Buffer to twice the bead volume and incubate for 30min with rotation at 4°C.

      - Wash once with RIPA Buffer and add RIPA Buffer to twice the bead volume.

      4. Elution and reverse cross-link

      4.1. Elute DNA by adding 120μl of Elution Buffer to the protein A/G beads and rotate for 15 min at 30°C.

      4.2. Spin down and transfer the supernatant into fresh tube.**

      4.3. The INPUTs can be included at this stage. Add 80μI of elution buffer (when using the DNA purification kit) or 400 μl TBS (when using phenol:chloroform) to each INPUT sample. Add either 2 μl RNase A (0.5 mg/ml) when purifying DNA using PCR purification kit or 5 μl of proteinase K (20 mg/ml) when purifying DNA using Phenol:Chlorophorm to the eluates (INPUTs and IP material) and heat at 65°C for 4-5hrs (or overnight).****

      4.4a.The DNA is then purified using a PCR purification kit following the manufacturer’s instructions.

      4.4b. Alternatively the DNA can be Phenol:Chlorophorm extracted and ethanol precipitated in presence of 10μl glycogen (5 mg/ml) and taken up in 100 μl H2O.

      4.5. Store samples at -20°C or proceed with detection method (PCR, microarray, etc).*****

      4.6. PCR is used to quantify DNA levels of specific loci. This is analyzed semi-quantitatively (analyses of PCR endproduct by agarose gel) using primers which can be designed using the URL below. http://biotools.umassmed.edu/bioapps/primer3_www.cgi

      Alternatively, DNA levels are quantitatively measured by real-time PCR. Primers and probes are often designed

      using software provided with the real-time PCR apparatus.

      **** The samples can be frozen and stored at -20°C after these steps.

      ***** The INPUT DNA is purified as described and the concentration should be calculated. Transfer 5 μl of the purified DNA in a tube containing 995 μl TE to give a 200-fold dilution and read the OD260. The concentration of DNA in μg/ml is OD260 x 10,000. This is used to calculate how much input DNA was included in the immunoprecipitation, and the sample values altered accordingly.

      Solutions

      FA Lysis Buffer

      50 mM HEPES-KOH pH7.5

      140 mM NaCl

      1 mM EDTA pH8

      1% Triton X-100

      0.1% Sodium Deoxycholate

      0.1% SDS

      Protease Inhibitors (add fresh each time)

      RIPA Buffer

      50 mM Tris-HCl pH8

      150 mM NaCl

      2 mM EDTA pH8

      1% NP-40

      0.5% Sodium Deoxycholate

      0.1% SDS

      Protease Inhibitors (add fresh each time)

      Wash Buffer

      0.1% SDS

      1% Triton X-100

      2 mM EDTA pH8

      150 mM NaCl

      20 mM Tris-HCl pH8

      Final Wash Buffer

      0.1% SDS

      1% Triton X-100

      2 mM EDTA pH8

      500 mM NaCl

      20 mM Tris-HCl pH8

      Elution Buffer

      1% SDS

      100mM NaHCO3

      Proteinase K

      Dissolve in H2O at 20 mg/ml, store at -20°C.

       

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