seeblue protein ladder mbd2 western blot

#SEEBLUE PROTEIN LADDER MBD2 WESTERN BLOT HOW TO#

Some smearing is expected, but if it is excessive, there may have been a problem with the gel electrophoresis step. If you see a reddish-pink smear where the bands should be, you may have a problem with your protein. Bands are smeared.Ī Ponceau S stain should produce crisp, discrete bands. Otherwise, check the transfer conditions, including the composition of the transfer buffer. If you are using PVDF, remember to activate the blot. If there is no Ponceau S-stained band after the transfer and the ladder is not visible on the blot, it is likely that your transfer did not work. Assuming the SDS-PAGE gel was stopped before the ladder and samples ran off the bottom of the gel, you should be able to see the location of the pre-stained protein ladder in the gel before you initiate the transfer to the membrane. The ladder is not visible on the membrane, and the Ponceau S stain shows no bands.Īgain, this shows the utility of a pre-stained ladder. You may not have homogenized the tissue sufficiently, or perhaps your cultured adherent cells were not dissociated from their plate. If you haven’t done a protein quantification assay to determine the protein concentration in your lysate, and there is no signal from Ponceau S staining, there is a chance your sample has very little or no protein. If the pre-stained ladder is visible, but there is no signal from the Ponceau S stain where your sample proteins should be, then it is reasonable to conclude that the problem lies not in the transfer but in the sample preparation. Using a pre-stained ladder in your SDS-PAGE can be very useful here. The ladder is visible on the membrane, but the Ponceau S stain shows no bands. 7 Problems With Your Ponceau S Staining and What it Means 1. However, Ponceau S is relatively insensitive compared to other alternatives, so it may not be for you if your sample contains less protein. Coomassie Blue and India Ink are incompatible with downstream applications. The advantage of Ponceau S is its simplicity and reversibility. There are alternatives, including Coomassie Blue and India Ink stains. Use standard lab PPE when working with Ponceau S as it can cause skin, eyes, and respiratory tract irritation. Is Ponceau S dangerous?Īs always, consult the material safety data sheet before using a new chemical. The results will be more accurate if you perform the staining before blocking. Since Ponceau S is a non-specific protein stain, it will also bind to blocking agents such as BSA or proteins in milk. Perform Ponceau S staining before the blocking step. Can I use Ponceau S stain after the blocking step? How should I dispose of Ponceau S after staining?ĭispose of the stain following your laboratory’s protocols. If you notice the strength is weaker, discard the old solution and replace it with a fresh solution. Generally, you can reuse Ponceau S until the signal strength is noticeably lower. The blocking step will remove any residual stain, and will not affect the experiment.įAQs about Ponceau S How many times can I reuse Ponceau S stain?

Proceed with blocking the membrane as usual.

Wash the membrane 3x with 5 mL of TBS-T or your washing buffer for western blot.

You may image the membrane at this point.

At this point, the protein bands stained with Ponceau S will be visible.

You can save money by reusing the used Ponceau S.

Rinse the membrane with distilled water until the background staining is removed.

Let the membrane incubate on the benchtop for a minimum of one minute.

Cover your membrane with the Ponceau S solution and close the lid to protect it from light.

After the transfer of proteins onto your membrane is complete, wash the membrane briefly with distilled water (one minute with rocking is sufficient).

Store the prepared solution at either 4☌ or room temperature, but ensure you protect it from light. The final concentration will be 5% v/v glacial acetic acid and 0.1% w/v Ponceau S. To make a 100 mL solution from the powder, weigh 100 mg of Ponceau S powder and make it up to 95 mL with distilled water before adding 5 mL of glacial acetic acid. Ponceau S can be purchased as a pre-made solution or as a dry powder.

#SEEBLUE PROTEIN LADDER MBD2 WESTERN BLOT HOW TO#

Read on to find out what these are! Ponceau S Staining Protocol How To Make a Ponceau S Solution If you’ve stained your blot and found a less-than-ideal pattern, it could be indicative of several issues. However, like anything else on the benchtop, things can sometimes go awry.

The staining process is typically done on a blot after the transfer step to confirm that the transfer worked well with no issues. It does not affect the protein structure.Three key benefits of Ponceau S stain to note: Example of Ponceau S staining of a western blot membrane.

Seeblue protein ladder mbd2 western blot