Nupage lds sample buffer manual

The power supply will maintain a constant voltage, according to how you set the knob. The current running through the gel will depend on the gel unit.

Set the display switch to milliamps to see the current. If the current is much lower or higher there may be a problem with the way the gel unit is set up; see the troubleshooting section at the bottom of this page. Also, as soon as you turn on the voltage, you should see bubbles being produced in the inner chamber due to water being split at the electrode. Once your gel is running, you should see the dye front quickly start to move down the gel.

This is the blue dye from the sample buffer. Your proteins aren't visible yet, but they will be migrating down the gel more slowly than the dye front. We're using gradient gels.

The top centimeter of the gel called the stacking gel is made with a low percentage of polyacrylamide to allow all the proteins to start migrating into the gel. The lower percentage has larger pores, which are ideal for separating high-molecular-weight proteins, while the higher percentage is best for smaller proteins.

There's no hard rule about how far to run your gel. Usually it's best to run it until the dye front gets close to the bottom of the gel; this takes about 50 minutes. If you run it too long, the dye front and the lower-molecular-weight proteins will eventually migrate out of the gel and disappear.

While you're waiting for the gel run to finish, fill a beaker with deionized water and warm it up in the microwave. You'll use this for washing the gel. Also, get a gel staining tray. When you're ready to stop, turn off the power supply and disconnect the leads, then remove the lid. Remove the gel cassette from the chamber and set it on a paper towel. Use a gel knife to separate the plates as shown below:. This is a delicate operation but it takes some force to break the plastic welds and separate the plates.

Start with the tall plate down and the short plate on top. Crack the plates apart all the way around, and you should be able to lift the short plate off. Make sure the gel isn't sticking to the short plate. Now you're ready to drop the gel into a gel staining tray filled with warm deionized water. The gel will tend to stick to the tall plate, because there is a "foot" of gel material sticking into the slit in the plate.

Use the gel knife to gently push the foot free of the plate. You won't be able to see any protein bands until you stain the gel.

Before you do that, you need to wash all the SDS out of the gel so the stain can bind to the proteins. Soak the gel in warm not hot deionized water for 3 minutes with occasional agitation. Repeat this process for a total of four 3-minute washes with warm water. Don't cut this short, or your gel won't stain well. You need to give the SDS time to diffuse out of the gel. You're using warm water to speed up diffusion. While you're doing the washes, clean up your gel unit , put it back together, and put it away.

You can throw away the comb and plates that came with gel, but don't throw away the buffer dam! That's part of the gel unit. Rinse all the parts of the gel unit briefly in deionized water, dry them carefully don't break the wires , and put everything back together for the next lab.

After the last wash, pour off all the water and pump some SafeStain on the gel. Use enough stain to fill the bottom of your tray and cover the gel. Cover the gel staining tray with plastic wrap and write your name and the date on the plastic.

Store the gel in the refrigerator until next time. You'll normally leave your gel in the stain from one lab period to the next. If the gel has been soaking in stain for days, the whole gel will be blue. You should be able to see the protein bands, but they will be partially obscured by the background of blue stain in the gel.

Destaining removes this background, leaving your gel transparent so the bands are easier to see. When you return to lab, pour the safestain into the labeled waste container, using the large funnel. Gently hold the gel in the tray with your gloved fingers. You'll probably have another lab activity to do on the same day, but start destaining the gel first. Pour some deionized water over the gel. Give it a quick rinse, pour the water down the sink, and add more water. Let the gel sit on your lab bench for a while, and you'll gradually see the background of the gel become more transparent, allowing your bands to stand out better.

Leave the gel in deionized water for an hour or more, changing the water every half hour. Meanwhile, you can get some other lab work done. Your gel should look more or less like this picture after a while -- strong blue bands against a clear background.

When you're ready, carry the gel tray over to the white light transilluminator. Place a piece of plastic wrap on top of the transilluminator and write your group name and the date on the plastic wrap, leaving room in the middle for the gel.

Lift the gel out of the water using a spatula or your fingers. Slide the gel onto the plastic wrap, and you're ready to take a picture. Article Title: Leucine zipper transcription factor-like 1 binds adaptor protein complex-1 and 2 and participates in trafficking of transferrin receptor 1 Journal: PLoS ONE doi: Article Title: The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of Paracoccidioides brasiliensis Journal: Molecular Microbiology doi: Figure Legend Snippet: A.

Pull-down assays to demonstrate that Gpa1 and Gpb1 both interact with Gpb1. Bound Gpa1 was detected as a gel band by autoradiography. Lanes 2, 4 and 6 establish that Gpa1 binds to immobilized Cyr1, but there was little difference in apparent affinity after incubation with GTP lane 2 , no nucleotide lane 4 or GDP lane 6. Negative controls, using immobilized GST, are shown in lanes 3, 5, 7, 10 and Each of the P.

WD1—7 and a series of truncates in which successive WD domains were deleted from the C-terminus e. WD2—7 ; a construct that comprised the two C-terminal WD domains e. WD6—7 ; and a fusion of WD domains 1 and 3 e.

As illustrated by the left-hand plate, full-length Gpb1 did not interact with Gpa1, Gpa2, Gpa3 nor Gpg1. The middle- and right-hand plates show the interaction of Gpg1 and Gpa1 respectively with the WD domains of Gpb1.

None of these control cells grew data not shown. Pull-down assays to demonstrate that Gpa1 interacts with Gpb1. Lanes 2, 4 and 6 establish that Gpa1 binds to immobilized Gpb1, but the apparent affinity decreases in order of incubation with GDP lane 2 , no nucleotide lane 4 and GTP lane 6.

Negative controls, using immobilized GST, are shown in lanes 3, 5, 7 and Use the gel immediately following the run. Do not soak the gel in running buffer After prying opening the gel cassette remove wells with the Gel Knife. May run some transfer buffer over surface to remove air bubbles. Place a piece of pre-soaked filter paper on top of the gel, and lay just above the slot in the bottom of the cassette, leaving the foot of the gel uncovered. Keep the filter paper saturated with the transfer buffer and remove all trapped air bubbles by gently rolling over the surface using a glass pipette as a roller Turn the plate over so the gel and filter paper are facing downwards over parafilm or clean flat surface Use the Gel Knife to push the foot out of the slot in the plate and the gel will peel off When the gel is on a flat surface, cut the foot off the gel with the gel knife Wet the surface of the gel with transfer buffer and position the pre-soaked transfer membrane shiny side down on the gel, ensuring all air bubbles have been removed Place another pre-soaked anode filter paper on top of the membrane.

Remove any trapped air bubbles Place two soaked blotting pads into the cathode - core of the blot module.

The cathode core is the deeper of the two cores and the corresponding electrode plate is a darker shade of gray. Carefully pick up the gel membrane assembly and place on blotting pad in the same sequence, such that the gel is closest to the cathode core follow manual Add enough pre-soaked blotting pads to rise to 0.

Blot Module to fit horizontally across the bottom of the unit. Push down gently on the knife handle to separate the plates. Remove the air bubbles by squeezing the pads while they are submerged in buffer.

Removing the air bubbles is essential as they can block the transfer of biomolecules if they are not removed. Use the gel immediately following the run. Do not soak the gel in running buffer After prying opening the gel cassette remove wells with the Gel Knife. May run some transfer buffer over surface to remove air bubbles. Place a piece of pre-soaked filter paper on top of the gel, and lay just above the slot in the bottom of the cassette, leaving the foot of the gel uncovered.

Keep the filter paper saturated with the transfer buffer and remove all trapped air bubbles by gently rolling over the surface using a glass pipette as a roller Turn the plate over so the gel and filter paper are facing downwards over parafilm or clean flat surface Use the Gel Knife to push the foot out of the slot in the plate and the gel will peel off When the gel is on a flat surface, cut the foot off the gel with the gel knife Wet the surface of the gel with transfer buffer and position the pre-soaked transfer membrane shiny side down on the gel, ensuring all air bubbles have been removed Place another pre-soaked anode filter paper on top of the membrane.

Remove any trapped air bubbles Place two soaked blotting pads into the cathode - core of the blot module.