Editing SDS-PAGE Gel Staining (section)

== Colloidal Coomassie ==

<br/>Colloidal Coomassie staining with CBB G-250<br/>1. Staining solutions<br/>Note: for the preparation of the staining solutions, use chemicals with high quality such as analytical grade (p.a.) and water of high purity as you get from Millipore systems (Milli-Q water).

{|
|-
| colspan="2" | 
Coomassie solution:

| ad 2000 ml H<sub>2</sub>O
|-
| 0.02&nbsp;% (w/v)
| CBB G-250
| 0,4 g
|-
| 5&nbsp;% (w/v)
| 
aluminum sulfate-(14-18)-hydrate

or ammonium sulfate

| 100 g
|-
| 10&nbsp;% (v/v)
| ethanol (96%)
| 200 ml
|-
| 2&nbsp;% (v/v)
| orthophosphoric acid (85&nbsp;%)
| 47 ml
|}

Note: for the preparation of the staining solution, the sequential addition of the components in the following order has to be maintained:

#first dissolve aluminum sulfate in Milli-Q water
#thereafter add ethanol, homogenize, and mix CBB G-250 to the solution
#as recently as the solution is completely dissolved, add phosphoric acid (the incorporation of the acid to the alcoholic media lets the Coomassie molecules aggregate into their colloidal state)
#finally fill up with Milli-Q water

Note: the final staining solution has a dark green-bluish appearance and is full of particles swimming around:

*Don't filtrate this solution!
*If you changed the order of preparation, you would get a more violet-bluish solution that is less sensitive.

{|
|-
| colspan="2" | Destaining solution:
| ad 2000 ml H<sub>2</sub>O
|-
| 10&nbsp;% (v/v)
| ethanol (96&nbsp;%)
| 200 ml
|-
| 2&nbsp;% (v/v)
| orthophosphoric acid (85&nbsp;%)
| 47 ml
|}

<br/>2. Staining procedure

*After second dimension separation, carefully remove the gels from the glass plates and transfer them into a staining dish filled with Milli-Q water.
*Wash the gels three times with Milli-Q water for 10 minutes on a horizontal shaker (insufficient washing causes poor sensitivity because the remaining SDS on the gels disturbs the bounding of the dye to the protein).
*Shake the Coomassie solution before use to disperse the colloidal particles evenly.
*Incubate the gels well covered with the Coomassie solution by agitation on a shaker for 2-12 hours. This staining generates marginal background so you can observe the staining progress in between.<br/>Note: after 10 minutes you can see first protein spots appearing, within 2 hours of incubation about 80% staining to its maximum level is completed. For nearly 100% staining, overnight incubation is recommended. In some cases, when the amount of protein to be stained is huge and the solution turns into a bright blue, a refresh of the staining solution is necessary.
*After the staining procedure remove the Coomassie solution and rinse the gels twice with Milli-Q water.<br/>Note: you can reuse the staining solution as long as particles are still remaining, otherwise discard it (keep in mind that your laboratory may have special regulations for waste disposal).
*Remove sticking dye particles from the staining dish with a lint-free paper towel and destain for 10- 60 minutes.<br/>Note: You can also wash the gels just with Milli-Q water but this we recommend only for preparative gels because there will last an irregular, weak Coomassie film on the gels that will arise during the scanning procedure.
*Finally rinse the gels twice with Milli-Q water: the gels will reach their original thickness (the alcohol-acid media makes the gels shrinking) and neutralization in water additionally enhances Coomassie's color intensity.



Colloidal CBB

Colloidal CBB staining came later on, thanks to the attempts to reduce the background staining. Diezel et al. observed that CBB-G in 12.5% Trichloroacetic acid is converted into a colloidal[23] hindering dye entry into the gels, therefore giving a low background. Neuhoff et al. was the first to develop a staining method using colloidal CBB-G in the presence of ammonium sulfate and phosphoric acid, demonstrating the benefits of adding alcohol to the staining solution.[24] This is allowed the detection of weak spots.[19,20] The addition of methanol in the classical staining solution (20% methanol, 1.6% phosphoric acid, 8% ammonium sulfate, 0.08% CBB G-250), intended to shift the equilibrium from the colloidal form to dispersed dye, and facilitated the staining;[24] however, this introduced a main critical point, as will be discussed below. The principal characteristics that make colloidal CBB an excellent staining method are its ease of use, extremely low signal-to-noise ratio and its sensitivity.[16] Colloidal particles are in equilibrium with dispersed dye in solution. Protein staining (and low background) is achieved when the free dye enters the gel, while the colloidal dye particles are excluded. Lately, Candiano et al.,[25] Wang et al. [26] and Pink et al. [27] proposed different protocols aiming to overcome the limit of detection of CBB staining, still requiring approximately 10 ng of protein in a band. 'Blue silver' was developed by Candiano et al. as a modification of Colloidal CBB increasing dye by 20% and phosphoric acid by fivefold (staining solution: 20% methanol; 10% phosphoric acid; 10% ammonium sulfate; 0.12% G-250).[25] The lower pH, causing protonation of Asp and Glu residues, is thought to promote more efficient hydrophobic binding to aromatic and hydrophobic residues, allowing a more intensive staining. Blue silver is more sensitive than the other colloidal CBB-G stains, with a detection limit approaching 1 ng for BSA. The possibility of destaining 'blue silver' exclusively with ultrapure water is also advantageous as it is low in cost and minimally affects stained bands. Another modified protocol was described by Wang et al.:[26] after fixing, staining solution employs 5% v/v acetic acid, 45% v/v ethanol and 0.125% (w/v) CBB R-250. Indeed, colloidal CBB-G staining is more sensitive than CBB-R in solvent solutions (8 ng).[14,18] The procedure requires multiple steps. Moreover, the need for two destaining solutions (first 5% v/v acetic acid, 40% v/v ethanol; then 3% v/v acetic acid, 30% v/v ethanol) renders the method prone to dye loss also by the stained proteins. Pink et al. [27] substituted acetic acid with phosphoric acid during the fixation (10%) and staining of the gels (8%) and also 5% aluminum sulfate (Al2(SO4)3) 14–18 hydrate in the staining solution. Al2(SO4)3 is used in the fabric industry as a mordant; however, Pink et al. stated that it also acts as an ion-bridging reagent, as Al3+ binds the dodecyl sulfate part of sodium dodecyl sulphate (SDS), by coagulation. Destaining is carried out in 2% phosphoric acid and 10% ethanol; afterwards gels are put in water overnight. Pink et al. modified this approach using a solution containing 10% ethanol, 0.02% of CBB G-250, and 8% phosphoric acid and 5% aluminum sulfate



colloidal CBB staining 0.02% CBB-G250 in 8% (w/v) phosphoric acid, 5% aluminum sulfate and 10% ethanol; for 3 h, after fixation 30% ethanol, 2% phosphoric acid; (C) CBB staining according to Neuhoff (gel was stained in 2% phosphoric acid, 10% ammonium sulfate, 20% methanol, 0.1% CBB G-250 overnight). CBB: Coomassie brilliant blue.