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Monoclonal IgM, Rat purification kit

Product code: RIKM-FF KIT

PRICE: 300 EUR/KIT

KIT CONTENT
(sufficient for 8 purifications with 100 ml of rat cell culture supernatant and/or ascites fluid/each)

  • Rat IgM Binding Gel (SepharoseTM fast flow) (Code : RIKM-FF) : 5 ml gel column.
    Binding capacity : approx. 12 mg Rat IgM/ml wet gel.
    Purity : 95% by SDS-PAGE.
    Maximum pressure : 3 bars (43 psi, 0.3 MPa).
    Gel life : approx. 50 cycles with routine regeneration.
  • Rat IgM Binding Buffer (Code : BBRM) 2x concentrated: 1,000 ml (add 1000 ml of distilled water before use).
  • Rat IgM Elution Buffer (Code : EBRM) 4x concentrated: 125 ml (add 375 ml of distilled water before use).
  • Rat IgM Precipitating Agent (Code : PARM) : 8 x 1 sachet of sufficient quantity for precipitating all IgM from 200 ml of rat cell culture supernatant and/or ascites fluid.

INSTRUCTION FOR USE

  1. Add with mild agitation 1 sachet of Precitating Agent (PARM) to 200 ml of cell culture spernatant (and/or ascites fluid) for 15 minutes. Stop the agitation and allows to stand for 30 minutes at 4°C. Centrifuge at 3000 g for 10 minutes. Discard the supernant from the pellet. Dissolve the pellet in 30 ml of rat IgM Binding Buffer (BBRM). Such a sample is ready to be loaded into the column.
  2. Equilibrate the column (RIKM-FF) with 20 ml of rat IgM Binding Buffer (BBRM). Set the valve to get a flow rate of approx. 30 ml/hour.
  3. Load the sample prepared in point 1 into the column prepared in point 2 at a flow rate of 30 ml/hour.
  4. Wash the column with 200 ml of rat IgM Binding Buffer (BBRM) at a flow rate of approx. 50 ml/hour.
  5. Elute the rat IgM with the rat IgM Elution Buffer (Code : EBRM) until the O.D. at 280nm of the eluent reaches the baseline level. Collect 10 fractions of 5 ml elution volume.
  6. Assay the elution fractions obtained as described in point 5, using the most appropriate system (SDS-PAGE, immunodiffusion, radioimmunoassay, Elisa...)
REGENERATION OF THE RAT IgM BINDING GEL
It is recommended to regenerate the gel after every 5 cycles of use.
  1. Wash the column with 10x volumes of NaOH 0.1M.
  2. Wash the column with 10x volumes of distilled water.
  3. Equilibrate the column 10x volumes of PBS (50 mM K2HPO4, 150mM NaCl) pH 7.4.
  4. Store the column at 4°C in the presence of NaN3 0.1% (w/v).
  5. For the next use, see INSTRUCTION FOR USE as described above.
If you need sterile materials, the regeneration can be carried out as follows.
STERILE REGENERATION OF THE RAT IgM BINDING GEL
(GEL SANITIZATION)
AFTER EVERY 5 CYCLES OF USE
  1. Wash 1 volume of gel column with 5 volumes of acetic acid 1 M.
  2. Wash this column with 10 volumes of sterile distilled water.
  3. Wash this column with 5 volumes of NaOH 1M.
  4. Wash this column with 10 volumes of sterile distilled water.
  5. Wash this column with 10 volumes of PBS (50 mM K2HPO4, 150mM NaCl) pH 7.4; NaN3 0.1%(w/v).
  6. The sterile gel column is now ready to be re-used.

  IgA, IgG, IgM, IgY Purification Kits