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Testing Rabbit Bleeds By Elisa【皇家赌场号hj85】

文章作者:生命科学 上传时间:2019-08-27

核心提示:You can test whether or not you have gotten an immune response to the peptide and how strong that immune response is

核心提示:You can test whether or not you have gotten an immune response to the peptide and how strong that immune response is by

核心提示:Materials:1) TS Buffer: 10 mM Tris pH 8.0150 mMMaterials:Testing Rabbit Bleeds By Elisa【皇家赌场号hj85】。1) TS Buffer: 10 mM Tris pH 8.0150 mM NaCl2) TST Buffer: TS Buffer with 0.05% Tween-203) Alkaline Phosphatase Buffer: 50 mM NaCarbonate1 mM MgCl2 4) AP Substrate: p-nitrophenyl phosphate . Sigma Cat.# N93895) AP-conjugated secondary Ab: AP-Rabbit anti-mouse IgG . Reconstitute in ddH2O as prescribed by manufacturer and aliquot and store at -70 °C.6) 100 mM EDTA pH 8.0Protocol:1) Add 50 µl of antigen to each well of the ELISA plate. I use a 1 µg/ml of purified Sec4p most antigens will work in the 1-10 µg/ml range, but you need to determine this emperically. Allow theantigen to bind to the wells at room temperature for 1-2 hours or overnight at 4 °C.2) Discard antigen solution by flicking into sink and then placing upside-down onto Kimwipes. Wash 1X with 100 µl TST Buffer. Add 200 µl TST with 2% BSA to block. Incubate at room emperature for 1 hour.3) Discard block solution and add 50 µl monoclonal cell culture supernatants directly or add the same volume of sera diluted in TST w/ 1% BSA. Incubate 1 hour at room temperature.4) Discard the primary antibody solution and wash 3X with TST.5) Add 50 µl of diluted secondary antibody conjugated to Alkaline Phosphatase. I use a 1:500 dilution of rabbit anti-mouse IgG . Incubate at room temperature for 1 hour. About 15 minbefore the end of the incubation start dissolving the p-nitrophenyl phosphate substrate tablets. For each ELISA plate dissolve 1 tablet in 5 ml of AP Buffer.6) Discard the secondary antibody solution and wash 3X with TST.7) Add 50 µl per well of the p-nitrophenyl phosphate solution and incubate at room temperature for 10-30 min. Stop the reactions by adding 50 µl of 0.1 M EDTA pH8.0. For screening monoclonals it is best to let them go until all of the positives are clearly visible, however if you want to be quantitative it is important not to let them get intensely yellow or they will be off scale--the plate reader is most accurate when the wells have just begun to clearly turn yellow.

You can test whether or not you have gotten an immune response to the peptide and how strong that immune response is by doing ELISAs against peptide conjugated to BSA. By conjugating to BSA, you will eliminate any signal for antibodies generated to KLH during immunization.

You can test whether or not you have gotten an immune response to the peptide and how strong that immune response is by doing ELISAs against peptide conjugated to BSA. By conjugating to BSA, you will eliminate any signal for antibodies generated to KLH during immunization.

Protocol:

Protocol:

A. Coupling Peptide to BSA

You need:

  • 1 ml of 2 mg/ml BSA in 0.1 M NaHCO3.
  • 1 ml of 0.2% glutaraldehyde in 0.1 M NaHCO3.
  • 0.5 ml peptide . Peptide can be added as a solid if soluble.
  1. Mix, adding glutaraldehyde last. Peptide and BSA turn a little yellow even before adding glutaraldehyde.
  2. Incubate 90' at 37 deg C.
  3. Add 0.1 volumes of 0.1 M NaBH4 in 0.1 M NaHCO3. There will be some bubbling. Add same amount of NaBH4 after 15'. Do a quick microfuge spin if there are too many bubbles.

A. Coupling Peptide to BSA

You need:

  • 1 ml of 2 mg/ml BSA in 0.1 M NaHCO3.
  • 1 ml of 0.2% glutaraldehyde in 0.1 M NaHCO3.
  • 0.5 ml peptide . Peptide can be added as a solid if soluble.
  1. Mix, adding glutaraldehyde last. Peptide and BSA turn a little yellow even before adding glutaraldehyde.
  2. Incubate 90' at 37 deg C.
  3. Add 0.1 volumes of 0.1 M NaBH4 in 0.1 M NaHCO3. There will be some bubbling. Add same amount of NaBH4 after 15'. Do a quick microfuge spin if there are too many bubbles.

B. ELISA with Peptide conjugated to BSA

  1. Coat 10 ug/ml antigen diluted in TBS ON at 4 deg C.
  2. Remove antigen and rinse wells 2Xs with TBST.
  3. Block 2 hr with 200 ul of 5% NFDM in TBST.
  4. Remove blocking reagent and rinse wells 2Xs with TBST.
  5. Incubate in primary antibody diluted in Blocking Buffer for 2 hr at RT. I do tripling dilutions beginning at 1/10 .
  6. Remove primary antibody and rinse wells 4Xs with TBST.
  7. Incubate in secondary antibody diluted in Blocking Buffer for 1 hr at RT .
  8. Remove secondary antibody and rinse wells 4Xs with TBST.
  9. Rinse wells 2Xs with 50 mM HCO3; 0.5 mM MgCl2, pH 10.
  10. Develop in 1 mg/ml p-Nitrophenyl phosphate in 50 mM HCO3; 0.5 mM MgCl2, pH 10 .
  11. Read A410 in ELISA reader.

B. ELISA with Peptide conjugated to BSA

  1. 皇家赌场号hj85,Coat 10 ug/ml antigen diluted in TBS ON at 4 deg C.
  2. Remove antigen and rinse wells 2Xs with TBST.
  3. Block 2 hr with 200 ul of 5% NFDM in TBST.
  4. Remove blocking reagent and rinse wells 2Xs with TBST.
  5. Incubate in primary antibody diluted in Blocking Buffer for 2 hr at RT. I do tripling dilutions beginning at 1/10 .
  6. Remove primary antibody and rinse wells 4Xs with TBST.
  7. Incubate in secondary antibody diluted in Blocking Buffer for 1 hr at RT .
  8. Remove secondary antibody and rinse wells 4Xs with TBST.
  9. Rinse wells 2Xs with 50 mM HCO3; 0.5 mM MgCl2, pH 10.
  10. Develop in 1 mg/ml p-Nitrophenyl phosphate in 50 mM HCO3; 0.5 mM MgCl2, pH 10 .
  11. Read A410 in ELISA reader.

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