By Markus R. Wenk
Biochemistry performs an incredible position in all components of the organic and clinical sciences. With many of the learn or prognosis all for those components being according to biochemically got observations, it truly is necessary to have a profile of good standardized protocols. This handbook is a easy consultant for all scholars, researchers and specialists in biochemistry, designed to assist readers in without delay taking off their experiments with no past wisdom of the protocol. The ebook dwells at the techniques utilized in designing the methodologies, thereby giving considerable room for researchers to change them in accordance with their learn necessities.
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2 Silver Staining In silver staining, the gel is Impregnated with soluble silver ions and developed by treatment with formaldehyde, which reduces silver ions to form an insoluble brown precipitate of metallic silver. This reduction is promoted by protein. Requirements (1) Fixative (200 ml): Mix 100 ml of methanol with 100 ml of water and 100 µl of formaldehyde. Formaldehyde is added freshly (2) Dithiothreitol (DTT) (1 mg/ ml): Dissolve 10 mg of DTT in 10 ml of water. Store as 1 ml aliquots at −20◦ C (3) 20% Silver Nitrate (25 ml): Dissolve 5 g of silver nitrate in 25 ml of water.
This technique can be applied to check for biochemical characteristics, such as post-translational modifications (phosphorylation or glycosylation), expression levels or interaction with other proteins (which may or may not be pulled down in a complex). In some cases, instead of analyzing the proteins on SDS-PAGE, they could be used for the enzymatic assays. Depending on the source of the primary antibody used, the choice of Protein A or Protein G is according to the species in which the primary antibody was raised.
8) Transfer the supernatant to a fresh tube. (9) Add 40 µl of 1N HCl and vortex for 15s. (10) Centrifuge at 14000 rpm for 2 min. (11) Collect the lower organic layer in a fresh tube and dry. (12) Store at −80◦ C. 3%NaCl (10:1) to the cell pellet. Vortex to make a cell suspension. Form an emulsion by shaking at 250 rpm for 1 hr at RT. 5 ml of Hexane. Emulsify again by shaking for 1 hr at 250 rpm RT. Centrifuge at 4000 rpm for 10 min to separate the phases. Transfer the upper phase into a fresh glass vial.