Beta-Actin Protein | Beta- Actin Western Blot (2023)

FAQs

Beta-Actin Protein | Beta- Actin Western Blot? ›

Beta-actin

Beta-actin, is usually used as a loading control for Western Blot to normalize the levels of protein detected by confirming that protein loading is the same across the gel.

These proteins play important roles in determining cell shape and controlling cell movement (motility). Studies suggest that β-actin may also be involved in relaying chemical signals within cells.

beta-actin – a housekeeping protein

Though beta-actin has a molecular weight of 42 kDa, degradation by apoptosis will produce 38-41 kDa fragments on a Western blot.

Beta Actin is one of six Isomers of the actin protein. It is a major cytoskeletal filament protein found in eukaryotes, prokaryotes and fungi.

beta- and gamma-nonmuscle actins differ by 4 amino acids at or near the N terminus and distant from polymerization interfaces. beta-Actin contains an Asp(1)-Asp(2)-Asp(3) and Val(10) whereas gamma-actin has a Glu(1)-Glu(2)-Glu(3) and Ile(10).

Protein molecular weight markers, sometimes referred to as protein standards or protein ladders, are used to estimate the molecular weight of proteins of interest and to monitor the progress of electrophoretic separation or transfer in Western blotting.

Beta-Actin (42 kDa) is commonly chosen as a loading control due to its general expression across all eukaryotic cell types. The expression levels of this protein do not vary drastically due to cellular treatment, which is another reason the protein makes a suitable control.

β-actin is a cytoskeletal protein involved in cell structure and motility. Based on these basic and ubiquitous cellular functions, GAPDH and β-actin are considered as housekeeping genes.

This β-actin mouse monoclonal primary antibody is used in a variety of assays and can be detected with IRDye anti-Mouse Secondary Antibodies.

Does western blot tell you how much protein? ›

Western Blots Are Semi-Quantitative

This is because they cannot tell you exactly how much of your protein of interest there is, only how much there is relative to another sample.

Loading too much protein leads to signal saturation in western blots, yet too little produces weak signals.

Western blot is often used in research to separate and identify proteins. In this technique a mixture of proteins is separated based on molecular weight, and thus by type, through gel electrophoresis. These results are then transferred to a membrane producing a band for each protein.

ACTB (Actin Beta) is a Protein Coding gene.

β-actin is a housekeeping gene and an obligatory part of the cell cytoskeleton. It expresses in almost all eukaryotic cells and is involved in controlling basic housekeeping functions such as development and maintenance of cell shape, cell migration, cell division, growth and signaling.

Our results confirmed the major cleaved sizes of β-actin were ∼27 kDa by granzyme B and were ∼32 kDa by caspase-3. Western blot analyses showed a 27-kDa band using antibody against N-terminal β-actin in the brains of 10-minute and 2-hour cerebral ischemia while no bands were detected for the caspase-3 product, fractin.

Radioactive materials that emit alpha and beta particles are most harmful when swallowed, inhaled, absorbed, or injected. Gamma rays are the most harmful external hazard. Beta particles can partially penetrate skin, causing “beta burns”. Alpha particles cannot penetrate intact skin.

Actin exists in two forms: G-actin (or globular actin) and F-actin (or fibrous actin).

The beta strands are parallel and hydrogen bonded to each other, while the alpha helices are all parallel to each other, and are anitparallel to the strands. Thus the helices form a layer packing against the sheet.

What is the difference between dot blot and western blot? ›

Dot blots are like Western blots in that they involve antibody-based detection of membrane-bound proteins. However, the main difference between the two techniques is that dot blotting does not require electrophoretic separation.

When presenting a western blot in a Starr lab meeting or presentation, include the following information: Title: Date, protein(s) and cell lysates including conditions being analyzed. Subtitle: Your initials, and date where details can be found in your lab book (see Lab Book Details).

Beta actin is commonly used as a western blotting loading control because it is ubiquitously expressed to high levels and is stable under most experimental conditions.

Loading controls can be used to show whether protein loading variation has occurred and may account for observed variations in the target band(s). When used correctly, loading controls ensure that proteins are quantitated appropriately despite subtle differences in loading amounts across all lanes of the Western blot.

What is a loading control? Loading Control: A loading control is an antibody specific for a ubiquitously and constitutively expressed protein and can be used to normalize protein levels in Western blot. Loading control antibodies help in assessing that samples have been loaded equally across a gel.

The most commonly used housekeeping gene-coded proteins are β-actin, β-tubulin and GAPDH. Those housekeeping proteins are used as internal controls in the western blot analysis with presumed stability and no changes in physiological condition.

β-actin, a traditional housekeeping gene, is widely used as an internal control to normalize gene and protein expression. However, β-actin expression can fluctuate upon the treatment with pharmacological agents or under some physiological and pathological conditions.

For example, GAPDH is more strongly expressed in the liver than beta-actin, but at lower levels in the arteries. Vice versa, beta-actin is more strongly expressed in the gizzard than GAPDH, but it is almost absent from cardiac muscle cells.

Western blot primary antibodies contain both monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs). In Western Blot, according to different purposes, different types of primary antibodies can be chosen.

m-IgG Fc BP-HRP, m-IgG₁ BP-HRP and m-IgGκ BP-HRP are the preferred secondary detection reagents for beta Actin Antibody (C4) for WB and IHC(P) applications.

What is beta actin antibody? ›

Beta actin antibodies provide a specific and useful tool for studying the intracellular distribution of beta actin and the static and dynamic aspects of the cytoskeleton. Because it is ubiquitously expressed in all eukaryotic cells, beta actin is frequently used as a loading control.

Can the protein marker be seen on the membrane? The prestained protein marker or ladder should be visible on the membrane after transfer. If not, then the transfer of the proteins from the electrophoresis gel to blotting membrane may have been unsuccessful.

Samples to be analyzed by western blot are denatured and reduced so that proteins resolve according to their molecular weight during electrophoresis. The addition of reducing agents, such as β-mercaptoethanol (BME) or dithiothreitol (DTT), reduces the disulfide bonds between cysteine residues.

Secondary antibody recognizes the primary antibody. Secondary antibodies used for western blotting are typically conjugated with an enzyme; the most commonly used enzymes are Horse Radish Peroxidase (HRP) and Alkaline Phosphatase (AP).

It may take up to 3 months before antibodies to HIV are produced after exposure. Since the Western blot test checks for antibodies, it may give false negatives for both conditions if it's administered too soon. In some instances, certain conditions such as lupus may also cause false positives to occur.

Amyloidosis is a condition in which too much of a particular protein (amyloid) collects in the organs, so that they are not able to work normally. Amyloidosis can affect the heart, kidneys, liver, spleen, nervous system, stomach or intestines.

Possible Causes

High blood protein levels are linked to several medical conditions and issues: Dehydration. Chronic (long-term) inflammation or inflammatory disorders. Viral infections such as hepatitis B, hepatitis C or HIV/AIDS.

The expression level of a gene can be calculated by measuring the transcribed mRNA (northern blot), the expressed protein (Western Blot), or by directly staining the protein or mRNA when it is still in the cell.

To improve western blot accuracy, samples should be quickly harvested in ice cold buffer (neutral pH) and should be immediately frozen in liquid nitrogen and stored at −80 °C until use to avoid proteolytic degradation by endogenous proteases (as sometimes indicated by gel streaks) [10].

β-Actin is expressed in all four chambers of the heart as well as in cultured adult cardiomyocytes [2], [3].

What are the 3 proteins in actin? ›

Other proteins (Arp2/3 proteins) can serve as nucleation sites to initiate the assembly of new filaments, so cofilin, profilin, and the Arp2/3 proteins (as well as other actin-binding proteins) can act together to promote the rapid turnover of actin filaments and remodeling of the actin cytoskeleton which is required ...

Beta-actin, is usually used as a loading control for Western Blot to normalize the levels of protein detected by confirming that protein loading is the same across the gel.

The sequence is a cDNA 1,866 bp in length and encodes a protein with 375 amino acids. This is similar to other animals.

The ACTB gene encodes beta-actin, which is essential for a number of cytoplasmic functions, such as regulation of cell shape and migration, as well as nuclear functions, such as regulation of gene expression, cell division, and proliferation (summary by Cuvertino et al., 2017).

In Western blotting, beta-actin (42 kDa) is commonly chosen as a loading control due to its broad and consistent expression across all eukaryotic cell types and the fact that expression levels of this protein are not affected by most experimental treatments (Figure 3).

The number or the 'band size' is a representation of your ribcage girth. We get this size by taking a measurement around your ribcage, about where the bra band sits. This measurement is taken in inches.

The amino acid sequence of skeletal and cardiac muscle actin consists of 375 amino acid residues, including one unusual amino acid residue, 3-methylhistidine, which is formed post-translationally.

beta-Actin Antibody from Cell Signaling Technology

This antibody recognizes Human, Mouse, Rat, Bovine, Drosophila/Arthropod, Hamster, Non-Human Primate, Zebrafish/Fish, and Other Mammalian antigen. The beta-Actin Antibody has been validated for the following applications: Simple Western, and Western Blot.

GAPDH is a key regulatory enzyme, which catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate during glycolysis. β-actin is a cytoskeletal protein involved in cell structure and motility. Based on these basic and ubiquitous cellular functions, GAPDH and β-actin are considered as housekeeping genes.

Beta-Actin (42 kDa) is commonly chosen as a loading control due to its general expression across all eukaryotic cell types. The expression levels of this protein do not vary drastically due to cellular treatment, which is another reason the protein makes a suitable control.

Why do you need a housekeeping gene for western blot? ›

The housekeeping proteins are used as reference proteins to normalize the target protein during western blotting analysis. Therefore, to accurately compare western blotting signals, one must compensate for these non-sample-related variations in signal intensity.

It is one of the so called housekeeping proteins and GAPDH is constitutively expressed in almost all tissues in high amounts. For this reason, GAPDH is widely used as a loading control for protein normalization in Western blotting.

To date, cancer researchers have predominantly used the GAPDH and β-actin reference genes as internal reference controls, because their mRNA expression levels are established to be high and constant in many different cells and tissues [4, 5].

GAPDH is frequently used as an endogenous control for quantitative RT-PCR analysis because its expression is consistent at different time points and various experimental manipulations [25].

β-actin is a housekeeping gene and an obligatory part of the cell cytoskeleton. It expresses in almost all eukaryotic cells and is involved in controlling basic housekeeping functions such as development and maintenance of cell shape, cell migration, cell division, growth and signaling.

ACTB (Actin Beta) is a Protein Coding gene.

Beta-actin is commonly used as a western blot loading control as is expressed within all eukaryotic cell types and is not affected by cellular treatments. Beta-actin is approximately 42 kDa and GADPH is approximately 36 kDa. They are used as a control as they remain stable in the presence of a wide range of compounds.

Actin exists in two forms: G-actin (or globular actin) and F-actin (or fibrous actin).