Question

Explain your observations on the staining of wool with Orange G and Methylene Blue at the pH 4, 7, and 9.

Discuss the factors that can influence protein/dye interactions at pH 4, 7, and 9.

Orange G structure

он SO3Na SO3Na

Methylene blue structure

Answer 1

Molecular Formula:	C16H18ClN3S
UNII:	8NAP7826UB
Chemical Names:	methylene blue 61-73-4 Methylthioninium chloride Basic blue 9 Swiss Blue

A compound consisting of dark green crystals or crystalline powder, having a bronze-like luster. Solutions in water or alcohol have a deep blue color. Methylene blue is used as a bacteriologic stain and as an indicator. It inhibits GUANYLATE CYCLASE, and has been used to treat cyanide poisoning and to lower levels of METHEMOGLOBIN.

Methylene Blue is a synthetic basic dye. Methylene blue stains to negatively charged cell components like nucleic acids; when administered in the lymphatic bed of a tumor during oncologic surgery, methylene blue may stain lymph nodes draining from the tumor, thereby aiding in the visual localization of tumor sentinel lymph nodes. When administered intravenously in low doses, this agent may convert methemoglobin to hemoglobin.

Methylene blue is an organic chloride salt having 3,7-bis(dimethylamino)phenothiazin-5-ium as the counterion. A commonly used dye that also exhibits antioxidant, antimalarial, antidepressant and cardioprotective properties. It has a role as an EC 1.4.3.4 (monoamine oxidase) inhibitor, an acid-base indicator, a fluorochrome, an antidepressant, a cardioprotective agent, an EC 3.1.1.8 (cholinesterase) inhibitor, a histological dye, an EC 4.6.1.2 (guanylate cyclase) inhibitor, an antioxidant, an antimicrobial agent, a neuroprotective agent, a physical tracer and an antimalarial. It contains a 3,7-bis(dimethylamino)phenothiazin-5-ium.          

Orange G or orange gelb

Orange G
он SO3Na SO3Na
Names
Other names Acid Orange 10 C.I. 16230
Identifiers
CAS Number	1936-15-8
3D model (JSmol)	Interactive image
ChEBI	CHEBI:82427
ChEMBL	ChEMBL410263 ChEMBL1615565
ChemSpider	10468647
ECHA InfoCard	100.016.096
KEGG	C19372
PubChem CID	9566064
CompTox Dashboard (EPA)	DTXSID6021082
InChI[show]
SMILES[show]
Properties
Chemical formula	C16H10N2Na2O7S2
Molar mass	452.38 g/mol
Hazards
Main hazards	R36/37/38, S26, S36
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
verify (what is ?)
Infobox references

Orange G or orange gelb^[1] is a synthetic azo dye used in histology in many staining formulations. It usually comes as a disodium salt. It has the appearance of orange crystals or powder.

Orange G can be used as a color marker to monitor the process of agarose gel electrophoresis, running approximately at the size of a 50 Base pair (bp) DNA molecule, and polyacrylamide gel electrophoresis. Bromophenol blue and xylene cyanol can also be used for this purpose. (However, the apparent "size" of all these dyes varies according to the concentration of agarose in the gel and the buffer system used, so one should look up the appropriate reference before using the dyes to determine how far a gel has run.)

the factors that can influence protein/dye interactions at pH 4, 7, and 9.

They are both migrating both towards the anode like nucleic acids. So they are easily spotable on a gel and depending on the condition of gel and migration they exhibit the mobility of DNA fragments of a certain size then you are able without staining the gel to estimate how far the samples have migrated.

Agarose and Metaphor Gels

1.Add agarose to 1X TBE (or TAE) buffer. For gel size 20 x 24 cm, use 300-400 ml buffer and 0.7 to 1.0% agarose.
2. Melt agarose in 500 ml flask in microwave oven, mixing several times during heating. Let cool to 55 C (until flask can be held).
3. Tape the ends of gel tray and place on a level bench.
4. Add ethidium bromide: 2.5 ul of 10 mg/ml stock per 100 ml. (gel cam also be stained in ethidium bromide bath after electrophoresis (see point 9).

NOTE: Ethidium bromide is mutagenic - wear gloves when handling, and use extra caution. Change gloves when contaminated and dispose in separate waste for ethidium bromide.

5. Pour agarose into tray and insert combs. Remove bubbles with a pipette tip. Allow to solidify.
6. Remove tape and place tray in gel boxes. Pour enough 1X TBE (or TAE) buffer into the gel box to cover the gel by at least 0.5 cm. Remove combs when ready to load samples.
7. Load 1 ug Lambda digested with Hind III (5 ul of 200 ng/ul stock) as molecular weight marker, then load samples.
8. Run at 15-25 V for 12-24 hours.
9. If no ethidium bromide was added to the agarose: stain gel in 1 ug/ml ethidium bromide (100 ul of 10 mg/ml ethidium bromide in 1000 ml dd H2O) for 20 min. 10. Rinse gel for 20 min in 1000 ml ddH2O.
11. Slide gel onto UV transilluminator and take photo.
Photographing tip:
Place small piece of paper with writing or transparent ruler on the gel to help focus.

MetaPhor® Agarose
High resolution agarose
Introduction
MetaPhor agarose is a high resolution agarose that challenges polyacrylamide. MetaPhor agarose is an intermediate melting temperature (75° C) agarose with twice the resolution capabilities of the finest-sieving agarose products. Using submarine gel electrophoresis, you can resolve PCR products and small DNA fragments that differ in size by 2%.


Analytical Specifications
Gelling temperature (3%) = 35° C
Melting temperature (3%) = 75° C
Gel strength (3%) = 300 g/cm²
Applications
• High resolution separation of 20-800 bp DNA fragments
• Recovery of fragments under 800 bp
• Fine analysis of PCR? products
• AMPFLP, STR and tri- and tetranucleotide repeat analysis

Suggested Agarose Concentrations

Final Agarose Concentration (%) Size Range (Base Pairs) 150-800 100-600 50-250 20-130 <80 1X TAE Buffer 2.0 3.0 4.0 5.0 1X TBE Buffer 1.8 2.0 3.0 4.0 5.0

Dye Mobility Table

Migration of double-stranded DNA in relation to Bromophenol Blue (BPB) and Xylene Cyanol (XC) in MetaPhor agarose gels.

1X TAE Buffer XC 480 200 120 85 1X TBE Buffer XC 310 140 85 60 0 0 BPB 70 40 35 30 Agarose 2.0 3.0 4.0 5.0 BPB 40 35 30 15

Precautions
Always wear eye protection when dissolving agarose and guard yourself and others against scalding solutions.

Microwave Instructions for Agarose Preparation
1. Choose a beaker that is 2-4 times the volume of the solution.
2. Add chilled 1X or 0.5X electrophoresis buffer and a stir bar to the beaker.
3. Slowly sprinkle in the agarose powder while the solution is rapidly stirred.
4. Remove the stir bar if not Teflon® coated.
5. Soak the agarose in the buffer for 15 minutes before heating. This reduces the tendency of the agarose solution to foam during heating.
6. Weigh the beaker and solution before heating.
7. Cover the beaker with plastic wrap.
8. Pierce a small hole in the plastic wrap for ventilation.

For agarose concentrations > 4%, the following additional steps will further help prevent the agarose solution from foaming during melting/dissolution:
a. Heat the beaker in the microwave oven on Medium power for 1 minute.
b. Remove the solution from the microwave.
c. Allow the solution to sit on the bench for 15 minutes.


9. Heat the beaker in the microwave oven on Medium power for 2 minutes.
10. Remove the beaker from the microwave oven. Caution: Any microwaved
solution may become superheated and foam over when agitated.
11. GENTLY swirl the beaker to resuspend any settled powder and gel pieces.
12. Reheat the beaker on HIGH power until the solution comes to a boil.
13. Hold at boiling point for 1 minute or until all of the particles are dissolved.
14. Remove the beaker from the microwave oven.
15. GENTLY swirl the beaker to thoroughly mix the agarose solution.
16. After dissolution, add sufficient hot distilled water to obtain the initial weight.
17. Mix thoroughly.
18. Cool the solution to 50-60°C prior to casting. Once the gel is cast, allow the molten agarose to cool and gel at room temperature. The gel must then be placed at 4° C for 20 minutes to obtain optimal resolution and gel handling characteristics.


Hot Plate Instructions for Agarose Preparation
1 . Choose a beaker that is 2-4 times the volume of the solution.
2. Add chilled electrophoresis buffer and a stir bar to the beaker.
3. Slowly sprinkle the agarose powder while the solution is rapidly stirred.
4. Weigh the beaker and solution before heating.
5. Cover the beaker with plastic wrap.
6. Pierce a small hole in the plastic wrap for ventilation.
7. Bring the solution to a boil while stirring.
8. Maintain gentle boiling until all the agarose is dissolved (approximately 10 minutes).
9. Add sufficient hot distilled water to obtain the initial weight.
10. Mix thoroughly.
11 . Cool the solution to 50-60°C prior to casting. Once the gel is cast, allow the molten agarose to cool and gel at room temperature. The gel must then be placed at 4° C for 20 minutes to obtain optimal resolution and gel handling characteristics.


Ordering Information:

Catalog No. 50-8 50-80-1 50-84 Size 25g 25g 500g

These products may be used as in vitro medical devices inaccordance with the U.S. Food, Drug, and Cosmetics Act.

As Dominique's said, uses of the two dyes are to track the DNA molecule in the gel during the course of gel electrophoresis. Generally, on a normal 0.8% or 1.0% Agarose gel, the bromophenol blue migration rate is equivalent to 350 - 400bp while Xylene cyanol is equivalent 3 - 4Kbp. So during electrophoresis, Xylene makes the lower dye front while bromophenol blue makes the upper dye front. These two dye front help the user to monitor the rate of migration and to prevent the over-running of gel