We have prepared a new method fast method for stainig SDS-PAGE with silver nitrate (1 hour duration) whis is bellow.

1) Fixation (20 mins)

12 ml acetic acid + 50 ml Methanol + 15 ul Formaldehyde (37%) complete to 100 ml with distiled water.

2) Washing (20 mins)

50 ml Ethanol+50 ml dd water

3) Washing with water (3x 20 seconds)

4) Silver Solution (15 mins)

0.1 gram silver nitrate+37 ul Formaldehyde (37%) complete to 100 ml with dd water

5) Revelation

2.5 grama Sodium Carbonate+25 ul Formaldehyde (37%) +100 ml dd water

keep the gel inside this solution till the band appear.

Disrupt the further staining with 50% acetic acid.

The Silver staining can stain nangrams of proteins while Coomassie can stain micrograms of proteins.

Silver-staining is another option. It is quite sensitive.

Silver-staining is a very good option!!!

If I'm impatient I microwave the gel in regular coomassie stain for ~45 sec. Put it in destain and nuke it again.

Silver staining is very sensitive, but not that fast.... All the "fast" silver based methods I tried so far have the same sensitivity of Colloidal Coomassie staining, and are by far more complicated...

I have used the microwave technique but got tired of avoiding the toxic methanol fumes and the destain step. With the Acqua stain you really do not need to destain the gel to see the bands which come up in several minutes.

We use Coomassie brilliant blue. Its the better than Silver. Because of the good contrast, high sensitivity and long term stability

I used Coomassie brilliant blue for rice root proteins and its having stability long term.

You may also try silver staining and microwave techniques.

we always using CBB strain, it is more sensitive and gives clear visible

I recommend to use the Mixture of CBB+ Methanol + Acetic Acid+ Water in shaker water bath for 30 mins and then followed by destaining using Destaining solution of Mixture of Methanol+ Acetic acid+water Overnight.

Next day will be the visualization day.

Dear Sir,

As many people mentioned, silver staining is better method. It's sensitive but will give good visibility of protein if it's in a very very small amount. But, the concentration of the gel plays a vital role here like 10%, 15% etc.,

All the best. Thank you.

Kind Regards,

Pradeep.

You can always use ethanol instead of methanol. Also put a wadded up Kimwipe tissue in the destain to soak up the free coomassie.

Coomassie blue stain

I go back and forth between Copper staining and Coomassie Brilliant Blue. Copper staining takes at most 5 minutes, and I destain with 3 washes diH2O for ~2 minutes. Copper staining doesn't fix your protein, and it can be reversed allowing you to blot or elute your protein.

I used this http://mybio.wikia.com/wiki/Rapid_ethanol-based_Coomassie_Blue_staining_of_SDS-polyacrylamide_gels

Generally Coomassie Brilliant Blue is good, and don't use too much protein concentration. Again use 10 & 12% SDS gel and dont run it too fast.

I would suggest you Comassie brilliant blue but if you need a more sensitive check then prefer silver staining.

Comassie brilliant blue

Usually cbb is used as staining dye for proteins. Alternatively u can use silver staining also. Sliver staining is highly sensitive staining method compared to cbb staining.

Silver staining can be done especially when the protein bands are light and not visible in other stain. It is rapid and effective. However, the amount of protein to be loaded per well needs to be standardized for best result.

What do you mean by "fast"? I could give you a protocol for Silver staining in 30 min, as it is at least 10-times more sensitive than Coomassie staining..

If using a Coomassie stain and if you want to decrease quickly and perfectly the backgroud add a laundry sheet in the washing solution !!!

I prefer Comassie brilliant blue. But for a fast and sensitive staining, you can try RAMA stain.... Here is the link: http://www.ncbi.nlm.nih.gov/pubmed/20503199

You can try PageBlue (CBB G-250 form Fermentas) and fast staining protocol with microwave. Staining is done in 40 min.

You can use 0.3 M ZnCl plus imidazole combination for Staing..refer this page for protocol and procedure http://camelot.bioc.cam.ac.uk/~marko/methods/zincimi.pdf

I tried this and its very fast and useful..

All the best.

I would recommend to you the silver staining, but this is not specific staining of proteins, in other words if your sample is not pure ,you are in risk to stain other biomolecules like polysaccharides, lipids,etc..

WE use Pierce (now Thermo Sci.) GelCode Blue for all kinds of gels. We soak the SDS gels in H2O for a% min, the into stain . Within an hour or less the band s become visible and the gel can be transferred to water for storage. I have never tried to dry gells. We scan them for a record and densitometry. PS For agarose gels we use Coomassie Blue G-250 (instead of R-250) in 10% acetic acid for o/n and destaining in 10% acetic acid.

Daniela Ross: Please give us that fast silver protocol - should be very useful if you have seen it fast and reliable. Is it the Nesterenko et al. protocol (J. biochem.biophys. methods 28:239-242 [1994]?, or that by Zhao et al. (Electrophoresis 33:2143 [2012])?, or what?

Hi Mary,

I have used GelCode Blue, and I like it but it requires the DI water washes (have tried to shorten/reduce them but loose sensitivity) and then it takes 15-30 min. at a minimum to see the bands clearly, I get the same sensitivity and results in 3-5 min. with the Acqua stain and no water washes!

There are many gel protein staining can use to stain the gel including bromophenol blue, Fast green , Amido black (Acjds Black 1) and coomassie blue also silver staining .

the silver stain is 100 times more sensitive for protein than the commonly used coomassie blue. for more reading:

Merril et al., 1984. in methods enzymology,

Dear Lars Andersson: After comparing, I can say that it is the 30 min protocol by Nesterenko et al. and it works nicely, but the protocol by Zhao et al. 2012 appears to be even more convenient (4 steps and 20 min).

I think silver stain is very effective and gives precise results.

Flamingo from Biorad is 10 times more sensitive than GelCode blue or Comassie Blue stain or silver staining. We can detect 5 ng protein staining on the gel.

Deep purple fluorescent stain is also very sensitive. Down to about 0.5 ng I think. Takes about an hour to stain, background is minimal.

Hi, you may want to try GeneScript commassie based gel blot staining, just 5 -6 min staining without destaining. It works as the iBlot from invitrogen by applying staining pack on the gel in sandwich format and click the start button, 6 min later, coommassie blue stained gel should be ready to be scanned.

Coumassie blue R-250 is a stain for routine work. There is a trick to make the destainig procedure fast and easy. Instead of time consuming destainng in acetic acid-methanol (ethanol) you may fix and destain by 3 x 3 min. washing in deionized(distilled) water in microwave oven at about 70-90 of Celcium. Coumassie G-250 is also useful.

The silver staining is much more sensitive, but more expencive and delicate procedure.

Comassie for sure

We have found the Imperial stain from Thermo to work very well for our applications.

Optiblot ab 119211 (abcam) - stains in minutes

Hi dear Gary

Selection of gel staining is dependent to other works that you intend to do on stained gels.

Silver staining is more sensitive but most MS analyser can not match with this staining. Also silver staining is approximatly expensive (because of silver nitrate'scost).

Commassie blue is simple and reliable staining method but it's sensitivity is low.

Another good method that simple but have high sensitivity (camparing to silver staining) is cloidal commassie staining.

If you need cloidal commassie staining protocole, please dont hesitate to contact me.

Cheers

Siver Stain from biorad is sensitive, easy to use, you could have good results in less than one hour.

Coomassie blue

BioSafe Coomassie from Biorad (G250) is a good choice. It's fast (results are obtained within 2 hours), there is no need for methanol/acetic acid (you just use water for destain) and is much more sensitive than standard coomassie (brilian blue R-250). If you need something even more sensitive, silver staining is the way (although less quantitative and usually not MS compatible)

Hey Gary,

I like Invitrogen, SimplyBlue SafeStain, easy to use.

Now I use GelCode Blue Stain Reagent (Thermo Scientific) instead Coomassie and I'm very happy. It's a fast and "smell-free" way. Plus washing with DI water (3x5 min) does not lead to protein degradation etc.

OK. I understood your problem. In our laboratory we use several differents staining methods to reveal proteins such as Coomassie blue R-350, G-250, in presence of ammonium sulfate, silver stainings - Morrissey, Blum, Bier and Gross methods. This latter is very sensitive showing low background). The fixative systems use methanol (30-40 %) better than ethanol.Sometimes TCA ( 5 -20%) + Sulfosalicil acid as fixative protein agents are also used and in some cases before do staining with CBR-350/ CBG-250. The use of TCA %is dependent of protein molecular weight. Recently we also used Sypro (Ruby) for protein fluorescence detection. Good luck for you.

Coomassie blue G-250........ 1g

Methanol ...................................1L

water ..........................................2.5L

mix well and use. 50 ml per gel and microwave for 1 min. stain for 5 min.

destain in

Acetic acid 10%

Methanol 30%

water to 1L

destain for 30 min to O/N

Your new stain sounds very attractive. Wouldn't you recommend it?

I use Instant Blue. It stains the gel in maximum 15minutes with no need to de-stain. Plus the background is really low. Also, it is a pretty sensitive. You can take a picture of your gel from anytime between 5-15mins. Later there is no harm to put it in water.

http://www.gentaur.com/fa.htm

Musa

yes I recommend the stain. it is inexpensive and good to use.

Cheers

Amruta, Instant Blue sounds a lot like Acqua blue.

This one is the best for daily uses...no smell, cheapest and very faaaaaaaaaast:-)

http://www.labtimes.org/labtimes/trick/tricks/2008_06.lasso

I use SYPRO Red (Lonza chemicals) stains to stain my gels. It takes only about 35 minutes to stain my gels with no destaining needed. However, I need to use a GE TYPHOON FLA 9000 system to visualize my gels.

Coomassie brilliant blue R-250 (Sigma-Aldrich). Washing depends how long you stain your gel. immersing gel in stain solution for 0.5 h at 370C is enough. prior to washing in destain, wash gel in tap water quickly and then destain on shaker. In two changes after every 15 min will bring the bands obvious.

We have prepared a new method fast method for stainig SDS-PAGE with silver nitrate (1 hour duration) whis is bellow.

1) Fixation (20 mins)

12 ml acetic acid + 50 ml Methanol + 15 ul Formaldehyde (37%) complete to 100 ml with distiled water.

2) Washing (20 mins)

50 ml Ethanol+50 ml dd water

3) Washing with water (3x 20 seconds)

4) Silver Solution (15 mins)

0.1 gram silver nitrate+37 ul Formaldehyde (37%) complete to 100 ml with dd water

5) Revelation

2.5 grama Sodium Carbonate+25 ul Formaldehyde (37%) +100 ml dd water

keep the gel inside this solution till the band appear.

Disrupt the further staining with 50% acetic acid.

The Silver staining can stain nangrams of proteins while Coomassie can stain micrograms of proteins.

I use SimplyBlueTM SafeStain (Invitrogen) with gentle shaking to stain my gels. Proteins stained using the SimplyBlue™ SafeStain are compatible with mass spectrometry (MS) analysis.

Selection of a staining method is dependent to cost, sensitivity, tools and time.

I recommend silver staining because it is very sensitive and need no specific tools.

If the cost is a problem for you, please use commassie brilliant blue staining.

On the other hand fluorescent detection of protein have good sensitivity but needs some specific tools that not found in all laboratories.

Silver staining is highly sensitive and best method to visualize proteins on gels.

We do silver staining protocol and could get crystal background clarity. In our 2-D gels we could see thousands of protein spots in the gel.

Instant Blue (Expedeon) is amazing, but it does take about 20min to get a nice report quality gel. no washes required.

Instant blue also, it's awesome. I use precast gels, take the gel from the plastics, put it directly in Instant blue, 15-20min you have you bands and then just water. Also it can be extensively reused!

Here is an awesome procedure that takes about 5 min with Simply Blue safe stain from Invitrogen.

1. Add water to gel, so that it is immersed. Heat in microwave for two minutes.

2. Discard water and add stain so that gel is covered. You can use Rubbermaid sandwich containers for the procedure.

3. Heat for 45 seconds. Let sit for two min.

4. Remove stain, and add water. Let destain. You can add tissue paper to hasten the destaining. Takes about 5 min. For good destaining picture quality gels, do the destain overnight.

For polyacrylamide sodium dodecyl sulphate (SDS-page) , I used coomassie brilliant blue R250 which prepared by dissolve the dye in methanol (0.1 g in 50 ml methanol) and add 10 ml acetic acid and 40 ml water )fresh preparation every time , this staining to protein with conc. (25-50 ul), if the your sample is less than 25 ul concentration you can silver staining with 0.1% concentration . also there are another stain like amido black 10B dye . (Ref. Manual on techniques in molecular biology (proteins)(1986). ; Peohling, H.M. and Neuhoff,V. (1981). Electrophoresis, 2, 141.

You don't need methanol to destain. If you are careful, you could destain the Coomassie blue out of the gel by putting the gel (just the gel, remove cassettes and anything that you have with the gel and DO NOT PUT FOIL with it) in water and in the microwave (a laboratory microwave in a hood, not a food microwave please) for a few seconds. Try increments of 5 or 10 seconds at a time so you don't dry your gel to a crisp. If it happens though, you might be able to hydrate it again by soaking it overnight in distilled water.

Here is a nice protocol with simple Blue.

1. Get a small sandwich Tupperware container.

2. Put gel in the container and add enough water to cover gel

3. Microwave 2 min and decant water

4. Add simple blue stain and microwave 45 sec. Wait to cool and decant

5. Add water and let destain for 5 min. Then take a picture

For real nice pictures, destain for longer.

Immerse your SDS-PAGE gel in chloroform or carbon tetrachloride for about 30 minutes, rinse with water, and watch on a UV transilluminator. Both these agents chemically modify the AROMATIC AMINO acids (particularly tryptophan) in proteins that then fluoresce under UV. You already know all the good ones by now..this one's archaic, not sensitive, and not useful but it's fun knowing!

Fermentas PageBlue works fin, you only stain for 60min, then you go directly to destain with water. Can reuse 10 times

AcquaStain from Bulldog Bio works well and is quite fast and sensitive

I tried InstaBlue and works Fine, 20 mins ready with no wash required

By looking at the application of the stained bands on SDS PAGE Slab, I would recommend a stain which can be easily removed like coomassie blue.

Try warming up destaining solution in a microwave briefly

you can try Silver Staining, you will find the information at

Blum et al., Electrophoresis, 8, 93 (1987).

Silver blue

Silver blue prepared at home as it is as sensitive as silver and easy to handle

Page Blue is fantastic

Problue safe stain from a small italian biotech, (named Giotto biotech), is methanol and acetic acid free. It is  a quite cheape safe comassie stainiin suitable for coloration in microwave.

 Generally i remove the gel, i briefly wash it with water,and stain it with problue for 5-10' in microwave (600W).  Afterwards i destaining the gel in milliQ water using again microwave... (2 wash -. 5 minutes at 1000W). The plastic box where you add the gels need to be partially closed to avoid complete stain or water evaporation and gel dryng.

In this way you can easly obtain a nice final coloration (see attached gel - 4-12% MES acquired with Gel doc EZ biorad) in less than 30 minutes.

http://www.giottobiotech.com/

I would suggest you a very simple and cost effective technique to visualize your bands in 10 mins. Follow the simple coomassie brilliant blue-R-250 staining protocol.

1. Staining solution: Note- Prepare fresh for fast staining (45% (v/v) methanol, 10% (v/v) acetic acid and 0.25% CBB-R-250). Dip your gel in this staining solution and heat the gel at about 65 degree celcius.

2. De-staining solution: (45% (v/v) methanol, 10% (v/v) acetic acid). After the staining is done, remove the staining solution and then add this de-staining solution to the gel and again heat it for 5 mins.

Cheers ;) you would surely be able to see your protein bands on the SDS-PAGE gel.

You can add a piece ofpaper towel or a filterpaper in the destain. this will adsorb all the dye as it comes out.

Coomasie brilliant blue can be used.

Coomasie brilliant blue and silver stain.

Look also for stain free detection: - as an example: "Application of MALDI-TOF-mass spectrometry to proteome analysis using stain-free gel electrophoresis".

Coomasie brilliant blue (CBB) and silver stains are used......But certainly it is required to look at stain free detection. 

i want to silver stain my 2D gels .my standard silver staining procedure failed to show any spots on my gels.is it possible to restain those gels again with silver staining?

It is impossible to restain the gels that have already been stained with silver.

You need to run and stain it again.

I never use Ag staniing. Sorry.

You could try Colloidal Coomassie G-250 from BioRad. It is a bit expensive, but it has some advantages, like low background, high sensitivity and superior reproducibility. Furthermore, it does not require addition of methanol or acetic acid, then it eliminates the need for hazardous waste disposal.

Try using 'SafeBlue protein stain' its quick easy and with in 10 min you can see the bands. No washingup nothing. works better without any background at all. Above all its not Coomassie.

You can please follow the links below to obtain certain valuable informations:-

1. https://www.thermofisher.com/in/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/protein-gel-stains.html

2. https://www.thermofisher.com/order/catalog/product/46628

3. http://camelot.bioc.cam.ac.uk/~marko/methods/zincimi.pdf

Also I found Expedeon's InstantBlue is being just am amazing! It's really just one step (no wash!!! no fixation!!!) And I can see the first results in about 5-10 minute! It's slightly expensive but you can get a good deal! And if you work with P32-labeled protein that would be indispensable for you!

Colloidal Coomassie G-250 method. 1 - gel washed 6,5 % HClO4, 10-15 min. 2 - gel soak in 6.5 % HClO4 contaned 100-150 mg/250 ml Coomassie-G250, 10-15 min. Both procedures perform at shaking by hands or on shaker. After second procedure gel placed in boiling (or near boiling point) water bath. After 5-10 min you can see results. Time gel washings can be reduced if gel thin or solutions fresh. Solutions may be used more than 10 times

To save the results I put the gel in clear file, remove the cloth drops of water and scans on an office scanner.

We  have used  GelCode Blue Stain Reagent [Product # 24592, Thermo Scientific]] for many years and for gels of all kinds. After washing briefly in water 2 times,  bands are visible in less than an hour, and then the gel is transferred to water.

Coomassie blue staining is based on the binding of the dye coomassie blue R250, which binds non-specifically to virtually all proteins.