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Monday, March 27, 2023

Simple Information to Repair 7 Western Blot Points

You understand how many issues can crop up in case you routinely carry out western blotting. Ponceau S staining can assist determine points early on, saving effort and time in incubating and growing unhealthy blots.

Learn on to find how Ponceau S works, how one can carry out staining, and get an in depth information to decoding what the stain means when troubleshooting your failed blots.

What Is Ponceau S and How Does it Work?

Ponceau S is a dye that binds to proteins on PVDF or nitrocellulose membranes, producing crimson bands the place there may be protein (Determine 1). In contrast to Coomassie Blue, the opposite normal dye for visualizing proteins after SDS-PAGE, Ponceau S is reversible and is, subsequently, suitable with most downstream functions.

Example of Ponceau S staining of a western blot membrane.
Determine 1. Instance of Ponceau S staining of a western blot membrane. (Picture Credit score: Thomas Warwick.)

Three key advantages of Ponceau S stain to notice:

  1. It doesn’t have an effect on the protein construction.
  2. It’s simply eluted after staining.
  3. It’s reusable.

The staining course of is often finished on a blot after the switch step to verify that the switch labored effectively with no points.

Nevertheless, like the rest on the benchtop, issues can generally go awry. In case you’ve stained your blot and located a less-than-ideal sample, it might be indicative of a number of points. Learn on to search out out what these are!

Ponceau S Staining Protocol

How To Make a Ponceau S Answer

Ponceau S might be bought as a pre-made resolution or as a dry powder. To make a 100 mL resolution from the powder, weigh 100 mg of Ponceau S powder and make it as much as 95 mL with distilled water earlier than including 5 mL of glacial acetic acid.

The ultimate focus might be 5% v/v glacial acetic acid and 0.1% w/v Ponceau S.

Retailer the ready resolution at both 4°C or room temperature, however make sure you defend it from gentle.

Staining Your Membrane With Ponceau S

  1. After the switch of proteins onto your membrane is full, wash the membrane briefly with distilled water (one minute with rocking is adequate).
  2. Cowl your membrane with the Ponceau S resolution and shut the lid to guard it from gentle.
  3. Let the membrane incubate on the benchtop for at least one minute.
  4. Rinse the membrane with distilled water till the background staining is eliminated. It can save you cash by reusing the used Ponceau S.
  5. At this level, the protein bands stained with Ponceau S might be seen. You might picture the membrane at this level.
  6. Wash the membrane 3x with 5 mL of TBS-T or your washing buffer for western blot.
  7. Proceed with blocking the membrane as traditional. The blocking step will take away any residual stain, and won’t have an effect on the experiment.

FAQs about Ponceau S

What number of occasions can I reuse Ponceau S stain?

It relies upon. Usually, you may reuse Ponceau S till the sign energy is noticeably decrease. In case you discover the energy is weaker, discard the outdated resolution and substitute it with a contemporary resolution.

How ought to I eliminate Ponceau S after staining?

Get rid of the stain following your laboratory’s protocols.

Can I take advantage of Ponceau S stain after the blocking step?

Carry out Ponceau S staining earlier than the blocking step. Since Ponceau S is a non-specific protein stain, it would additionally bind to blocking brokers akin to BSA or proteins in milk. The outcomes might be extra correct in case you carry out the staining earlier than blocking.

Is Ponceau S harmful?

As at all times, seek the advice of the fabric security knowledge sheet earlier than utilizing a brand new chemical. Use normal lab PPE when working with Ponceau S as it might trigger pores and skin, eyes, and respiratory tract irritation.

Are there options to Ponceau S?

There are options, together with Coomassie Blue and India Ink stains. The benefit of Ponceau S is its simplicity and reversibility. Coomassie Blue and India Ink are incompatible with downstream functions.

Nevertheless, Ponceau S is comparatively insensitive in comparison with different options, so it might not be for you in case your pattern incorporates much less protein.

7 Issues With Your Ponceau S Staining and What it Means

1. The ladder is seen on the membrane, however the Ponceau S stain exhibits no bands.

Utilizing a pre-stained ladder in your SDS-PAGE might be very helpful right here. If the pre-stained ladder is seen, however there isn’t a sign from the Ponceau S stain the place your pattern proteins must be, then it’s affordable to conclude that the issue lies not within the switch however within the pattern preparation.

In case you haven’t finished a protein quantification assay to find out the protein focus in your lysate, and there’s no sign from Ponceau S staining, there’s a probability your pattern has little or no or no protein. You might not have homogenized the tissue sufficiently, or maybe your cultured adherent cells weren’t dissociated from their plate.

2. The ladder isn’t seen on the membrane, and the Ponceau S stain exhibits no bands.

Once more, this exhibits the utility of a pre-stained ladder. Assuming the SDS-PAGE gel was stopped earlier than the ladder and samples ran off the underside of the gel, you need to be capable of see the placement of the pre-stained protein ladder within the gel earlier than you provoke the switch to the membrane.

If there isn’t a Ponceau S-stained band after the switch and the ladder isn’t seen on the blot, it’s seemingly that your switch didn’t work. In case you are utilizing PVDF, bear in mind to activate the blot. In any other case, verify the switch situations, together with the composition of the switch buffer.

3. Bands are smeared.

A Ponceau S stain ought to produce crisp, discrete bands. In case you see a reddish-pink smear the place the bands must be, you might have an issue together with your protein.

Some smearing is anticipated, however whether it is extreme, there might have been an issue with the gel electrophoresis step. There might be a number of culprits for this one:

  • Use contemporary 2-mercaptoethanol within the pattern loading buffer to make sure any disulfide bonds are damaged.
  • Guarantee you might be utilizing adequate SDS within the pattern loading buffer, the operating buffer, and the gel itself.
  • Make sure the glycine focus is excessive sufficient within the pattern loading buffer to allow correct stacking of the pattern earlier than getting into the separating gel.
  • In case you are uncertain about any buffers, make them contemporary and check out once more. This typically resolves the issue.
  • If the smeared bands are particularly massive and outstanding, you have got seemingly overloaded protein. Scale back the quantity of pattern loaded.

4. There is just one huge band close to the highest of every lane.

This means that the protein separation by electrophoresis was unsuccessful. When you have a purified pattern, your acrylamide gel pore measurement is likely to be too is just too small in your protein of curiosity. Strive making a gel with a decrease share. Sometimes, a gel must be lower than 10% if the protein analyte is bigger than 80 kDa.

In case you are utilizing an entire cell or tissue lysate, the issue lies with the separation itself. Make sure that the pH of the operating buffer and tris options used to make the gel are correct, and verify the electrophoresis settings to make sure the voltage is excessive sufficient to maneuver the proteins by way of the gel.

5. The stain is inconsistent, or there’s a house that’s not staining.

There’s a downside together with your switch. Guarantee there aren’t any air bubbles between the membrane and the gel when establishing the sandwich, and take care when inserting the sandwich into the equipment.

Forcing the sandwich into the equipment might trigger the gel to interrupt, the membrane to wrinkle, or enable house for an air bubble to intrude with the switch (Determine 2).

An example of poor western blot transfer visualized using Ponceau S staining
Determine 2. An instance of poor western blot switch visualized utilizing Ponceau S staining. (Picture credit score: Thomas Warwick.)

6. The stain is stronger in some lanes and weaker in others.

This isn’t essentially an issue together with your method, but it surely exhibits a unique quantity of protein between lanes. In case you want even protein focus throughout lanes, attempt a Bradford or BCA assay earlier than loading your samples to find out the precise portions to load.

The quantity of protein added is extra necessary than the quantity of the answer right here, however you want to bear in mind the dilution of your loading buffer and add the suitable quantity.

7. The Ponceau S stain seems nice, however I don’t see any bands after my antibody incubations!

For the reason that Ponceau S stain seems good, you may safely say that the issues seen on the finish of the process don’t stem from the electrophoresis or switch steps. Have a look at your blocking, washing, antibody incubations, and substrate visualization steps to find out what occurred.

Bonus Ponceau S Staining Suggestions

Don’t neglect to verify the sign on each the highest and backside of the blot. The proteins don’t switch equally to either side, so a weak or strange-looking sign might imply that you simply’re trying on the improper aspect of the blot.

It sounds apparent, however generally the obvious solutions are the final ones we consider within the lab (particularly whenever you’re operating on much less sleep than is good and also you actually want this blot to work)!

Moreover, western blots might be irritating, and generally issues don’t work out. In case you’ve tried every little thing and your Ponceau S stains are nonetheless trying irregular, generally the very best factor to do is figure on one thing else as we speak and check out with a stage head from scratch the following day. The reply could also be proper there, and also you would possibly must step away from it for some time.

Ponceau S Staining Summarized

Western blots can go sideways at any step within the protocol. Having a ‘checkpoint’ close to the halfway level of the protocol can assist slender down the place any points stem from. Ponceau S staining can assist you accomplish this.

Relying in your staining patterns, the issue might lie together with your pattern preparation, the electrophoresis, or the switch.

Alternatively, if the Ponceau S stain seems good, however the western blot nonetheless isn’t working, then you realize to troubleshoot the latter a part of the process from the blocking step onwards.

Ponceau S staining is usually a useful step in your western blotting protocol to make sure every little thing goes in keeping with plan. Hopefully, after studying this text, you need to use this method to scrub up your blots and get higher outcomes! Have you ever bought different tips about utilizing Ponceau S staining? Depart a remark under.

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