Experimental Tips | Doing This Step Could Boost Your Western Blot Success Rate by at Least 50%!

As the saying goes, "A good start is half the battle!" Today, we're diving into the crucial first step before beginning your experiment—getting to know your target protein!

The unique characteristics of your target protein can directly or indirectly influence your experimental outcomes. Here, we'll guide you through several key aspects, including protein localization, expression patterns, and post-translational modifications, to help you better understand your target and achieve clear, beautiful Western blot bands!

How Target Protein Characteristics Influence Western Blot Experiments

Part 1: Tissue Localization

Determining your target protein’s tissue localization is the first step in choosing suitable samples for Western blot experiments.

Database for reference: https://www.uniprot.org/
For example, when searching for human proteins,

you may find that the target protein is expressed in the heart, liver, skeletal muscle, and strongly expressed in skeletal and tongue smooth muscles. Based on this information, you can select the most appropriate tissue samples according to your specific experimental requirements.

Part 2: Subcellular Localization
Identifying the subcellular localization of your target protein is crucial for selecting the appropriate lysis buffer. If, for instance, the protein is localized in the plasma membrane, lipid rafts, or membranes, you’ll know exactly which lysis buffer to choose. 

Additional tips:

Always confirm the target protein’s subcellular localization from the databases or literature.
Choose appropriate extraction reagents based on subcellular localization for optimal WB results.

Part 3: Loading Controls in WB Experiments
A loading control antibody typically recognizes proteins encoded by housekeeping genes with relatively stable expression across mammalian tissues and cells. They serve as reference markers for evaluating the relative expression levels of target proteins.

Common loading control antibodies include:
Cytoplasmic markers: β-actin, GAPDH, β-tubulin

Nuclear markers: Lamin B1, HDAC, PCNA

These proteins maintain stable expression across different tissues and conditions and serve as reliable controls during protein quantification.

Impact of Target Protein Expression on WB Results

The difference in protein expression levels significantly affects experimental outcomes. Here's how to handle commonly encountered scenarios of varying expression levels:

For proteins with low expression, choose tissues or cells with higher expression levels (as previously introduced).

For proteins requiring induction (such as inflammatory pathway proteins), stimulation is necessary to detect expression clearly.

Example:

c-Fos expression is typically undetectable in untreated cells. It can be induced by serum starvation followed by treatment with 20% FBS for 2 hours, or with 200 nM PMA for 4 hours.

Western Blot example:

 

Lane 1: RAW264.7 cell lysate (untreated)

Lane 2: RAW264.7 serum-starved for 16h, then treated with 200 nM PMA for 4h

You will notice an evident difference between unstimulated and stimulated conditions, clearly highlighting the importance of induction conditions.

Impact of Post-translational Modifications (PTM) on WB

Post-translational modifications (PTM) refer to chemical alterations occurring after protein translation. Common PTMs include glycosylation, phosphorylation, methylation, acetylation, and ubiquitination, significantly affecting protein folding, stability, localization, and function. Glycosylation primarily impacts protein stability, folding, distribution, and activity. There are three main types: N-linked glycosylation, O-glycosylation, and glypiation. Phosphorylation mainly occurs on Serine (Ser), Threonine (Thr), or Tyrosine (Tyr) residues, widely studied for its role in signaling and regulation. Ubiquitination tags proteins for degradation through the proteasome pathway.

Possible effects of PTM on Western Blot:

PTMs can result in actual band sizes differing from theoretical predictions. For instance: Glycosylation may significantly increase the molecular weight compared to predicted values.
Phosphorylation or other modifications could also cause molecular weight shifts, resulting in discrepancies between theoretical and observed molecular weights.

Glycosylation can cause protein bands to appear heavier than their predicted molecular weight.

Phosphorylation can create distinct bands at higher molecular weights due to shifts in mobility on gels.

Tips

Besides target proteins, antibody selection is crucial for successful Western blotting. HUABIO offers high-quality, rigorously validated antibodies, ensuring reliable performance and consistent results, effectively assisting researchers in accelerating their scientific endeavors.