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●●●●●　Micro-protein assay　●●●●●



This micro-protein assay is not as accurate as other methods for assaying protein concentration, but it has the distinct advantage of requiring only trace amounts (less than 1ug) of your valuable protein. In many cases the accuracy (within a factor of 2) of the assay is more than sufficient. Another advantage of the assay is its relative ease and quickness.


  1. Materials

    1. Amido black stain
      0.25% Amido black 0.625g
      45% MeOH 112.5ml
      45% ddH2O 112.5ml
      10% glacial HOAc 25ml

    2. Amido black destain
      same as the stain without the dye

    3. Cellulose acetate dissolving solution
      80ml formc acid
      10ml glacial HOAc
      1ml 100% TCA

  2. Methods

    1. Starting with 5mg/ml BSA (in saline), make a series of 1:2 dilutions in saline (100ul BSA + 100ul saline). 8 - 10 dilutions is fine.
    2. In duplicate, spot 1ul of each BSA dilution, and the samples to be tested to a strip of cellulose acetate.
    3. Allow the spots to air-dry. If the samples contain alot of sucrose or glycerol. Re-wet the spot with 2ul aliquots of ddH2O & allow to dry again. Repeat this 3 - 5 times.
    4. Stain in amido black stain for 10 minutes, then destain in several transfers of destain until the background is nearly white.
    5. Examine the intensities of the black spots. Compare the spots from your test protein with those of the standards to obtain an estimate of your protein concentration.
    6. If a more accurate estimate is required:
    7. Carefully punch out one of each duplicate spot & dissolve in 400ul dissolving solution
    8. Measure the A523 of each sample. Plot the BSA standards as a standard curve & interpolate the concentration of the test samples using this curve.

Michael Thomm, personal communication
JWBrown, unpublished data

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