Technique Lab Report

Results:
The protein expression was done by the lab officers. However, I found a flowchart that can
represent the workflow for the protein expression.
Image created by Felicia Vulcu and Vivian Leong. Centrifuge clipart obtained, with
permission from Presenter Media, © 2009-2021 Eclipse Digital Imaging, Inc.
Protein extraction and clarification steps:
The colour of the protein after protein extraction is light yellowish with some white pellets.
Protein purification steps:
1. Collect 50 microlit of supernatant and store in tube labelled S

2. Collect 50 microlit of supernatant and store in tube labelled P
3. Wash histrap with 3 mL of water
4. Follow up with 5 mL of Binding Buffer
5. Add all of the supernatant to the column
6. Collect 1 mL → Flow Through (FT)
7. Wash with 5 mL of Binding Buffer, collecting the first 3 mL in 3 different
microcentrifuge tubes (1 mL each)
8. Label microcentrifuge tubes → W1, W2, W3 (W = wash)
9. Run 5 mL of Elution buffer, collect first 2 mL → E1, E2 (E = Elution)
10. Discard last 3 mL
After protein purification, the colour of it is light yellow, translucent.
Discussion:
By inserting a recombinant DNA plasmid into the host E. coli cell, the process of protein
expression allows for the synthesis of a protein that does not exist in nature (heterologous
protein). A DNA fragment that encodes the target protein you're aiming to create is included
in the recombinant DNA plasmid. A vector (or plasmid) harbouring the foreign gene and the
host cell are the two key elements in this procedure.
The process of separating and purifying protein from samples of whole tissue, cell cultures,
or biological fluids is known as protein extraction. The assay's starting material and
objectives are taken into account while designing the protein extraction technique. When
creating a protein extraction technique, it is crucial to keep the experiment's objectives in
mind because some buffer options (such as high salt, high detergent formulations) might
sabotage an experiment when higher order protein structure and function must be
conserved. Starting with extremely unclean materials, the protein extraction procedure
cleans them up using filtration, centrifugation, solubilization, and precipitation before
polishing them using methods like affinity columns and immunoprecipitation.
Protein purification is a critical step in the analysis of individual proteins, protein complexes,
and the discovery of interactions with DNA, RNA, and other proteins. There are several
different protein purification techniques to handle required scale, throughput, and
downstream applications. Often, the best course of action must be discovered empirically.
The most effective protein purification procedure is dependent on the protein being purified
as well as a number of additional variables, such as the cell used to generate the
recombinant protein (e.g., prokaryotic versus eukaryotic cells). Due to its simplicity of use,
quick cell growth, and affordable culture, Escherichia coli continues to be a top option among
researchers for the production of recombinant proteins. Relatively large amounts of proteins
synthesised in E. coli can be purified, although these proteins, particularly eukaryotic
proteins, might not show appropriate protein function or folding. Affinity purification tags may
be attached to a target recombinant protein to streamline the purification process (Nilsson et
al. 1997). Polypeptides, tiny proteins, or enzymes are frequently used as fusion tags and are
attached to a recombinant protein's N- or C-terminus. Different tags' biochemical
characteristics affect how well-behaved, soluble, and expressed the proteins to which they
are linked (Stevens et al. 2001). Recombinant protein purification is facilitated by using
expression vectors with fusion tags.
Conclusion:
Samples collected during:

Expression: Before induction (BI)
Extraction: Supernatant (S), Pellet (P)
Purification: Wash 1 (W1), Wash 2 (W2), Wash 3 (W3), Elution 1 (E1), Elution 2 (E2)
These are the samples collected and stored for the next lab. The next step would be to use
these samples to detect recombinant turnip yellow mosaic virus core (TYMVc) antigen via
SDS-PAGE and Western blotting.

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Results:

Protein extraction and clarification steps:

1. Collect 50 microlit of supernatant and store in tube labelled S

After protein purification, the colour of it is light yellow, translucent.

Samples collected during:

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