Micromeritics

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A handbook of Micromeritics





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Cleaning Validation | Campaign Cleaning Validation (CCV)

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When the product demand does not allow for a complete clean up after each batch, a campaign cleaning validation must be conducted.   A back to back cleaning is performed between batches in a campaign, and a complete cleaning at the end of a campaign. Sampling of the equipment is performed after the last batch in a campaign, and all test results must meet predetermined acceptance criteria as per campaign cleaning validation protocol.   Samples for residual active may be taken after back to back cleanings where possible, for information only . Microbiology samples may be taken after back to back cleanings in a campaign to ensure that there is no excessive microbial growth on the equipment.
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Bradford Protein Assay for determining protein concentration

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Principle: The Bradford protein assay is used to measure the concentration of total protein in a sample. The principle of this assay is that the binding of protein molecules to Coomassie dye under acidic conditions results in a color change from brown to blue. This method actually measures the presence of the basic amino acid residues, arginine, lysine and histidine, which contributes to formation of the protein-dye complex. Unlike the BCA assay, reducing agents ( i.e. DTT and beta—mercaptoethanol) and metal chelators ( i.e. EDTA, EGTA) at low concentration do not cause interference. However, the presence of SDS even at low concentrations can interfere with protein-dye binding. Materials and Reagents Bovine Serum Abumin (BSA) (Sigma-Aldrich) Coomassie Brilliant Blue G-250 (Sigma-Aldrich, catalog number: 27815 ) Methanol Phosphoric acid (H3PO4) Bradford reagent (see Recipes) Equipment Spectrophotometer (Tecan) Whatman #1 paper (Whatman) Procedure ...
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Western blot | Re-use a primary antibody

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Why reuse primary antibody? Western blotting is one of the most common method for detecting and semi-quantifying specific proteins. The cost of running a Western is predominantly driven by detection antibodies, which are used to probe membrane-bound target protein(s). Although some antibodies can be produced cheaply in-house, most antibodies must be commercially ordered. Reusing antibodies for subsequent Westerns can save your lab a considerable amount of money. So money is nothing but everything. In conclusion economic research practises is  budget friendly and reuse of primary antibody is one of them.  When reuse is problematic? The most standard phospho-antibody protocols involve blocking with 5% milk in PBS or TBS (0.1% Tween 20), followed by a couple washing steps with PBS or TBS to remove the excess milk.  If you dilute your antibody in a milk solution, storage and re-use will be extremely problematic, because the milk will "go bad" and fall...
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