β-Actin | β-Actin Western Blot (2023)

FAQs

What is the purpose of β actin on the western blot? ›

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.

To know how to analyze western blot data, Look for the sizes of the bands. These will be represented by a number, either followed by “kDa” or preceded by “p.” This is the size of the protein which has been detected and is the scale on which the proteins are separated in a Western blot.

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.

Single cell (fibre) Western blotting demonstrates the ability to detect proteins in small sample sizes, 5–10 μg total mass (1–3 μg total protein).

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.

beta-Actin has been frequently used as an internal control (gene) or as a housekeeping gene to normalize the expression of the target gene(s) or mRNA levels between different samples.

There are two types of western blot test that are used, an IgM western blot and an IgG western blot. These are separate tests that look for two different antibodies. Most of the time, the IgG western blot must be positive in order to indicate a potential infection with Lyme disease.

The Consortium for Retrovirus Serology Standardization (CRSS) has defined a positive test result as the presence of either p24 or p31, plus a diffuse envelope band (i.e., gp41 or gp120/160) (14).

The Western blot test separates the blood proteins and detects the specific proteins (called HIV antibodies) that indicate an HIV infection. The Western blot is used to confirm a positive ELISA, and the combined tests are 99.9% accurate.

The ACTB gene provides instructions for making a protein called beta (β)-actin, which is part of the actin protein family. Proteins in this family are organized into a network of fibers called the actin cytoskeleton, which makes up the structural framework inside cells.

Is it beta actin or B actin? ›

Actin beta (HUGO Gene Nomenclature Committee abbreviation ACTB/ACTB) is one of six different actin isoforms which have been identified in humans. This is one of the two nonmuscle cytoskeletal actins. Actins are highly conserved proteins that are involved in cell motility, structure and integrity.

The bound antibodies are then detected by developing the film. As the antibodies only bind to the protein of interest, only one band should be visible. The thickness of the band corresponds to the amount of protein present; thus doing a standard can indicate the amount of protein present.

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

I prepared protein samples from different mice tissues, however, I wonder how much amount should I load the samples on the SDA-PAGE gel for western blotting? For total protein extracted from cell or tussle lysate, 10-40 μg is recommended; for purified protein, 10-100 ng is recommended.

Protein extract should not be too diluted to avoid loss of protein and large volumes of samples to be loaded onto gels. The minimum recommended concentration is 0.1 mg/mL, optimal concentration is 1–5 mg/mL).

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.

To normalize expression, multiply the density of the target protein in each lane by the ratio of loading control density from the control sample (lane 1) to the loading control density of other lanes.

Western Blot Image Analysis: Volume Box Tools

One of the most intuitive ways to quantify a band is to simply draw a box around it and quantify the signal within the box.

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.

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.

What is the primary antibody for beta-actin? ›

β-Actin Antibody detects endogenous levels of β-actin. Due to the high sequence identity between the actin isoforms, β-Actin Antibody may cross-react with other actin isoforms.

False reactivity on the EIA or Western Blot assays can be due to HLA antibody, autoimmune diseases (such as lupus), cross reactivity to yeast, or to other contaminating antigens used to prepare the HIV antigens.

An IgG immunoblot result is considered positive if any 5 of 10 bands (the 18-, 23-, 28-, 30-, 39-, 41-, 45-, 58-, 66-, and 93-kDa bands, with the 23-kDa band representing OspC) are present (10).

The same Western blot patterns that are detectable in early seroconversion can also be seen in persons with false-positive results due to cross-reactivity with an epitope on gp41, which can give rise to multiple Western blot Env bands and a positive Western blot interpretation.

Positive and negative controls in western blots. The blue box indicates the positive control, where a known protein band is expected to appear and contain the target protein. The red box indicates the negative control, where the target protein is not expected (Image Source: LI-COR).

Second, the IgM Western Blot test result is only meaningful during the first four weeks of illness. If you have been infected for longer than 4 to 6 weeks and the IgG Western Blot is still negative, it is highly likely that the IgM result is incorrect (e.g., a false positive).

Each darker line on a column represents an area of binding by an antibody to a specific antigen. This Western blot assay is used to diagnose many infectious diseases, including Lyme disease. It is important to note that these tests do not directly detect the presence of the B. burgdorferi spirochete.

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.

Multiple nonspecific bands on the blot may be due to antibodies of poor quality or at too high a concentration, insufficient blocking, or nonspecific binding due to the presence of SDS.

ACTB (Actin Beta) is a Protein Coding gene.

What are the three types of actin? ›

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

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.

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 N-terminus of actin contains between two negative charges (yeast actin) and four negative charges (mammalian and avian muscle actins). Neither the myosin loop 2 (Rayment et al.

No bands are visible on the blotting 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.

Too much acrylamide in the gel. The bands may be very high on the blot if there's too much acrylamide in the buffer. This is because a high acrylamide density can block effective migration of proteins through the gel. You should generally run higher molecular weight proteins with a lower percentage of acrylamide.

Observation: Faint Bands (Weak Signal)

Antibody may have low affinity to protein of interest. Increase antibody concentration (2-4 fold higher than recommended starting concentration). Increase the amount of total protein loaded on gel. Mix enzyme and substrate in a tube.

Eating too much protein for a prolonged period of time can place a burden on the kidneys, liver and bones, as well as potentially increase the risk for heart disease and cancer.

But one common downside of going high with the protein is that it can also leave you a bit, ahem, stuffed up. Yup, we're talking constipation, diarrhea, and other not-so-pleasant gastrointestinal issues.

What happens if you transfer a western blot too long? ›

If you are transferring too long, you will detect protein on the second membrane as well. This is more likely to occur to the smallest proteins first so pay particular attention to where the lower molecular weight ladder bands should appear.

Protein Free Blocking Agents

PVP is a water-soluble polymer that binds to nitrocellulose and PVDF membranes. PVP is generally used at 0.5-2% concentration and is commonly combined with purified casein or other blocking agents.

Load all samples into gel lanes starting with the MW standards. Sample loading volumes should be from 5 µL–35 µL per lane (depending on gel). If protein concentrations are from 100 µg/mL–500 µg/mL,then sample amounts will range from 0.5 µg–17.5 µg per lane.

For generating whole cell lysates, we recommend using Cell Lysis Buffer, RIPA Buffer, or SDS. Cell Lysis Buffer is great because it can be used in a variety of applications and it does not contain harsh detergents that will denature your proteins. RIPA Buffer contains NP40 and sodium deoxycholate.

We recommend sample loads between 1 and 10 μg of protein per well. Optimize primary and secondary antibody dilutions. In most cases, this means reducing the total amount of antibody used (higher dilutions). This will limit the amount of HRP in the system to prevent saturation of signal.

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.

RIPA (radioimmunoprecipitation assay buffer) – This buffer is ideally used for whole cell extracts, membrane-bound proteins, and nuclear proteins. The RIPA buffer is compatible with most applications (e.g., protein assays and other protein purification techniques).

There are a number of different types of actin with slightly different structures and functions. α-actin is found exclusively in muscle fibres, while β- and γ-actin are found in other cells. As the latter types have a high turnover rate the majority of them are found outside permanent structures.

β-Actin forms the bundles of microfilaments which are connected to focal adhesions, whereas γ-actin is localized in a dense network (Dugina et al., 2009).

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.

What is the difference between alpha and beta releases? ›

Alpha testing is the first phase of formal testing, during which the software is tested internally using white-box techniques. Beta testing is the next phase, in which the software is tested by a larger group of users, typically outside of the organization that developed it.

Current gain α is defined as the ratio of collector current to the emitter current at constant collector voltage. Current gain β is defined as the ratio of the collector current to the base current at constant collector voltage.

The production of smooth muscle or actin antibodies is strongly associated with autoimmune hepatitis. It may also sometimes be seen in other forms of liver disease, such as primary biliary cholangitis (PBC), but usually at lower antibody titers.

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.

Beta actin and its mRNA are localized at the plasma membrane and the regions of moving cytoplasm during the cellular response to injury. J Cell Biol. 1991 Feb;112(4):653-64.

We have assigned six members of the human beta-actin multigene family to specific human chromosomes. The functional gene, ACTB, is located on human chromosome 7, and the other assigned beta-actin-related sequences are dispersed over at least four different chromosomes including one locus assigned to the X chromosome.

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.