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Western blot/Tissue lysate preparation

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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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Blending | Effective Sampling During Process Validation | Optimize Sampling Location

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Important of sampling during blending: Sampling plays a great role in achieving the accurate results of analysis. Sampling must and procedure must be defined in validation protocols and training should be provided to the concerned staff before the validation activities start. Samples are representative of the whole batch. So it should be handled and weight carefully because segregation may occur during weighing and transpiration. Sample quantity should not be more than the required and whole mass sample should be used in the analysis. Fig: Blending Machine Sampling for blend: Blends are tested for their homogeneity. Homogeneity of the blend is critical for the quality of the final product. The sample should be taken from at least 10 locations in the blender. The sampling location should be selected according to the difficulty of the blending. Ares of poor blending must be covered in sampling. Corners and discharged point must have...
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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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