BIOTECHNOLOGY
In Fig. 5 the NQO1 (11451-1-AP) enzyme serves as a quinone reductase together with conjugation reactions of the hydroquinones involved in detoxification pathways, as well as in biosynthetic processes such as the vitamin K-dependent gamma- carboxylation of glutamate residues in prothrombin synthesis. NQO1 has three isoforms: 26, 27 and 31 kDa MW, and the formation of homodimers (66-70 kDa) is needed for its enzymatic activity. Mlx-interacting protein (MLXIP,
also known as MONDOA) (13614- 1-AP) acts as a transcription factor, forming a heterodimer with MLX protein. Tis complex binds and activates transcription from CACGTG E boxes, playing a role in the transcriptional activation of the glycolytic target and glucose-responsive gene regulation. MLXIP has three isoforms: 110, 57 and 69 kDa, and the molecular weight of the MLXIP-MLX heterodimer is 130 kDa.
PROTEIN ISOFORMS Many proteins encoded by a single gene exist in more than one sequence variant, or protein isoform, due to alternative splicing during mRNA maturation. Tis can result in additional protein-coding sequences and higher molecular weight protein products, or proteins of lower molecular weight owing to premature stop codons. In addition, some proteins have multiple translation start sites, which give rise to isoforms with different N-termini. Protein isoforms can have differing half-life and subcellular localisation, may interact with diverse subsets of proteins, form distinctive protein complexes, and may have altered, even opposite, functions.
TECHNICAL OBSTACLES Antibody cross-reactivity. It is possible for the selected antibody to recognise not only its target protein, but also to cross-react non-specifically with other proteins in the analysed sample. Protocol optimisation and the implementation of an appropriate controls panel can help to minimise these issues.
Suggested controls may include: • Positive controls - purified target protein
- lysate from a cell line known to express the target protein
- lysate from a cell line overexpressing the target protein
• Negative controls - lysates from cell lines with lower expression of the target protein
- lysates from cell lines with the target protein knocked down (e.g. by siRNA or shRNA) or knocked out (e.g. by CRISPR)
Experimental optimisation can be achieved by adjusting one or a combination of the following: • extraction buffers (e.g. RIPA buffer) • blocking buffers (e.g. 5% skimmed milk or BSA)
• incubation and washing times (e.g. overnight at 4°C or 1.5h at room temperature (RT))
• secondary antibodies used for detection (e.g. dilution factor)
• membrane type (nitrocellulose vs. PVDF).
NON-SPECIFIC PROTEOLYTIC CLEAVAGE AND PROTEIN DEGRADATION Proteins can undergo non-specific proteolytic digestion if the protein sample is not handled correctly. Proteases released during cell lysis or tissue extraction can then cause protein fragmentation, resulting in smaller fragments of lower molecular weights being run on the Western blot. Some proteins are more susceptible to degradation than others, and consequently the choice of cell/ tissue lysis buffers and lysis conditions, along with supplementation with protease inhibitors, are vital for efficient protein extraction. Tis article has described the most commonly encountered reasons for observed discrepancies in the molecular weight of examined proteins. Identifying these will not only help with analysis of Western blotting results, but can also provide valuable insight into protein function, and physiology of studied biological processes.
Dr Karolina Szczesna is with Proteintech.
wwwpgtlab.com
Fig. 4
Fig. 5
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