What is a loading control and why is it used?

What is a loading control and why is it used?

A loading control is a protein used as a control in a Western blotting experiment. Typically, loading controls are proteins with high and ubiquitous expression, such as beta-actin or GADPH. They are used to make sure that the protein has been loaded equally across all wells.

What do loading controls do?

Loading controls are antibodies that are used to detect proteins within samples. When western blots are used to determine the levels of protein expression in a sample, loading controls ensure that the results aren’t due to loading or protein transfer errors.

What is the purpose of a loading control in Western blot list two common loading controls and explain why they are used as loading controls?

Loading controls serve numerous purposes in a Western blot investigation. They are essentially used to normalize the levels of protein detected within a sample by ensuring that protein loading is the same across the gel.

What is a good loading control for Western blot?

Therefore GAPDH, together with beta actin and tubulin is one of the most commonly used loading controls. We offer alpha tubulin and GAPDH DyLight® 680, DyLight® 750 and DyLight® 800 conjugated antibodies which are perfect as loading controls in fluorescent Western blotting experiments.

Is vinculin a good loading control?

Another cytoskeletal protein, vinculin, has been used as a Western blot loading control as well. It can be used as a loading control for high molecular weight proteins. In muscle tissues, a splice variant with an extra exon with the molecular weight of 150 kDa is also expressed.

Why is tubulin used as a loading control?

Beta-Tubulin, is usually used as loading control for Western Blot to normalize the levels of protein detected by confirming that protein loading is the same across the gel. OriGene offers high quality Anti-beta actin mouse mAb (Loading control) for Western Blot. View all beta-tubulin loading control antibodies.

How much protein should I load in a western blot?

Make sure you load at least 20–30 µg protein per lane, use protease inhibitors, and run the recommended positive control. Use an enrichment step to maximize the signal (eg prepare nuclear lysates for a nuclear protein). Overuse of antibodies has reduced their effectiveness.

Why is actin used as a loading control?

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.

How much protein should I load for a well?

How Much to Load. Consider the concentration of your purified protein, lysate, or culture sample. Ideally, it is best to load ≤2 µg per well of a purified protein or ≤20 µg of a complex mixture like whole cell lysates if you are doing Coomassie stain only.

How do you dilute protein for Western blotting?

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). Centrifuge for 20 min at 12,000 rpm at 4°C in a microcentrifuge.

When does a load control system pay for itself?

Many report that a load control system can pay for itself in a single season. When the decision is made to curtail load, it is done so on the basis of system reliability. The utility in a sense “owns the switch” and sheds loads only when the stability or reliability of the electrical distribution system is threatened.

How does load control work in a distribution network?

In a distribution network outfitted with load control, these devices are outfitted with communicating controllers that can run a program that limits the duty cycle of the equipment under control. Consumers are usually rewarded for participating in the load control program by paying a reduced rate for energy.

Which is the most common form of load control?

Ripple control. Ripple control is the most common form of load control, and is used in many countries around the world, including Australia, New Zealand, the United Kingdom, Germany, the Netherlands, and South Africa.

When did the first load management system start?

Modern utility load management began about 1938, using ripple control. By 1948 ripple control was a practical system in wide use. The Czechs first used ripple control in the 1950s. Early transmitters were low power, compared to modern systems, only 50 kilovolt-amps.