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Preparation of Epoxides03:00

Preparation of Epoxides

9.4K
Overview
Epoxides result from alkene oxidation, which can be achieved by a) air, b) peroxy acids, c) hypochlorous acids, and d) halohydrin cyclization.
Epoxidation with Peroxy Acids
Epoxidation of alkenes via oxidation with peroxy acids involves the conversion of a carbon–carbon double bond to an epoxide using the oxidizing agent meta-chloroperoxybenzoic acid, commonly known as MCPBA. Since the O–O bond of peroxy acids is very weak, the addition of electrophilic oxygen of peroxy acids to...
9.4K
Preparation of Amides01:29

Preparation of Amides

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Amides are synthesized by treating carboxylic acids with amines in the presence of dehydrating agents like dicyclohexylcarbodiimide (DCC).
The DCC-promoted synthesis of amides begins with the protonation of DCC by carboxylic acid. The protonation makes it a better acceptor. Next, the addition of carboxylate to the protonated carbodiimide gives a reactive acylating agent.
Subsequently, the amine acts as a nucleophile that attacks the acylating agent to form a tetrahedral intermediate. In the...
4.1K
Preparation of Nitriles01:12

Preparation of Nitriles

2.7K
One of the common methods to prepare nitriles is the dehydration of amides. This method requires strong dehydrating agents like phosphorous pentoxide or boiling acetic anhydride for converting amides to nitriles. Another reagent namely, thionyl chloride also accomplishes the dehydration of amides, where amide acts as a nucleophile. The first step of the mechanism involves the nucleophilic attack by the amide on the thionyl chloride to form an intermediate. In the next step, the electron pairs...
2.7K
Preparation and Reactions of Thiols02:33

Preparation and Reactions of Thiols

7.6K
Thiols are prepared using the hydrosulfide anion as a nucleophile in a nucleophilic substitution reaction with alkyl halides. For instance, bromobutane reacts with sodium hydrosulfide to give butanethiol.
7.6K
Preparation of Acid Anhydrides01:07

Preparation of Acid Anhydrides

4.0K
One of the methods for preparing symmetrical or unsymmetrical acid anhydrides involves the treatment of acid chlorides with the sodium salt of carboxylic acids. The reaction proceeds via a nucleophilic acyl substitution.
The carboxylate ion acts as a nucleophile that attacks the carbonyl carbon of the acid chloride to form a tetrahedral intermediate. Subsequently, the re-formation of the carbonyl group with the loss of the chloride ion as a leaving group leads to the formation of an acid...
4.0K
Preparation and Reactions of Sulfides02:26

Preparation and Reactions of Sulfides

5.8K
Sulfides are the sulfur analog of ethers, just as thiols are the sulfur analog of alcohol. Like ethers, sulfides also consist of two hydrocarbon groups bonded to the central sulfur atom. Depending upon the type of groups present, sulfides can be symmetrical or asymmetrical. Symmetrical sulfides can be prepared via an SN2 reaction between 2 equivalents of an alkyl halide and one equivalent of sodium sulfide.
5.8K

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Related Experiment Video

Updated: Feb 8, 2026

Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples
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Preparing Whole-Cell Lysates for Immunoblotting.

Larisa Litovchick

    Cold Spring Harbor Protocols
    |July 4, 2018
    PubMed
    Summary

    This protocol details protein sample preparation for immunoblotting using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer for efficient cell lysis. This method is effective for protein analysis but disrupts native protein structures, limiting its use in other applications.

    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Cell Biology

    Background:

    • Protein sample preparation is crucial for various biological analyses, including immunoblotting.
    • Solubilizing proteins requires specific buffers, salts, and detergents compatible with downstream procedures like gel electrophoresis.
    • Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer is commonly used for protein extraction.

    Purpose of the Study:

    • To describe a protocol for preparing protein samples for immunoblotting.
    • To highlight the use of SDS-PAGE sample loading buffer for efficient protein solubilization.
    • To identify the limitations of this lysis technique for applications requiring native protein structure.

    Main Methods:

    • Adherent cells are cultured in tissue culture dishes.

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    Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates
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  • Proteins are solubilized using SDS-PAGE sample loading buffer.
  • The protocol focuses on protein extraction for immunoblotting analysis.

    • Main Results:

    • SDS-PAGE sample loading buffer rapidly and efficiently extracts most cellular proteins due to the strong anionic detergent, SDS.
    • SDS denatures proteins and disrupts protein-protein interactions.
    • The lysis technique is detrimental to applications requiring intact protein structures.

    Conclusions:

    • SDS-PAGE sample loading buffer is effective for protein extraction for immunoblotting.
    • This method is unsuitable for applications such as enzymatic activity assays or protein-protein interaction studies.
    • Researchers must consider the impact of SDS on protein structure for specific experimental needs.