Submitting Proteomics Analysis Samples

Please contact us before submitting any samples. You can contact us by email, phone (404-778-4589), or fax (404-778-4281).

Also see the sections below for more information on sample submittal for specific services.

Please discuss the project with facility personnel prior to sample submission.

Sample Hazardous Materials letter for Customs

"Contaminating" Proteins. Keratins and their fragments (typical MW range: 40 kD-80 kD) originate from airborne dust in most laboratory environments and from gels, reagents and solvents. Calf serum albumin and its fragments (MW range: 35 kD-68 kD) and transferrin often originate from tissue culture media. Heavy and light chains of IgG contaminate immunoprecipitated proteins. Immobilized proteins (affinants, baits) from resins such as Sepharose contaminate selectively desorbed proteins. Other abundant proteins (eg, heat shock proteins) may also be present but not relevant. The value of running proper controls can not be overestimated.

Sample and data storage and retrieval Due to space limitations, all samples will be discarded three (3) months after reporting the results unless you request (mcflab@emory.edu) that they be returned. For sample returns (via Federal Express), we request that appropriate mailing information and a FedEx account to be billed are provided. Due to data storage limitations, all electronically acquired data will be stored for twelve (12) months after the final report. Hard copies of the reports will be stored for three (3) fiscal years after completion of the project.

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Placing Orders

Billing information

Proper billing information must accompany sample submission including the PI’s name, address, phone and fax numbers and e-mail. A correct billing address and P.O./Account # are also required. We will not begin work on any sample without an account number. For questions regarding billing, please contact Kelly Gassett at (404) 778-5932 or kgasset@emory.edu.

Oligonucleotide Synthesis

There are several ways in which to place a Oligonucleotide Synthesis order with our facility:

• Use our on-line order form
• Download Adobe Acrobat format (*.pdf) order form and fax it to us at 404.778.4281.
• siRNA - Download Adobe Acrobat format (*.pdf) order form and fax it to us at 404.778.4281

If you have any questions or wish us to purify a previously synthesized oligonucleotide please contact us.

Peptide Synthesis and Modifications

There are several ways in which to place a Peptide Synthesis order with our facility:

• Use our on-line order form
• Download Adobe Acrobat format (*.pdf) order form and fax it to us at 404.778.4281.

If you have any questions or wish us to modify/purify a previously synthesized peptide please contact us.

Protein/Peptide Sequencing

If you wish to place a internal digest order, please contact us first.

• Download Adobe Acrobat format (*.pdf) order form and fax it to us at 404.778.4281.

Internal Digestion of Proteins and Peptide Mass Fingerprinting (PMF)

If you wish to place a internal digest order, please contact us first.

• Download Adobe Acrobat format (*.pdf) order form and fax it to us at 404.778.4281
• If you have any questions please contact us.
• ICAT (SRP): Download Adobe Acrobat format (*.pdf) order form and fax it to us at 404.778.4281

Other Services

Please contact us first regarding any special services which you would like us to perform

 

• Mass Spectrometry: Download Adobe Acrobat format (*.pdf) order form and fax it to us at 404.778.4281
• Amino Acid Analysis: Download Adobe Acrobat format (*.pdf) order form and fax it to us at 404.778.4281
• Peptide/Protein Purification Request: Download Adobe Acrobat format (*.pdf) order form and fax it to us at 404.778.4281
• Ion Exchange Chromatography: Download Adobe Acrobat format (*.pdf) order form and fax it to us at 404.778.4281
• Special Research Project: Download Adobe Acrobat format (*.pdf) order form and fax it to us at 404.778.4281

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Protocols

Listed below are some of the protocols which we use in our lab. Please feel free to examine them and use them. Give us a call if you have any questions. Please see our chart regarding minimal protein amounts needed. (*.pdf)

Staining of gels and PVDF membranes ranked by decreasing order of sensitivity

• Gels
  • Silver Staining
  • RUBY GEL Staining
  • GelCode Blue Staining
  • Coomassie Staining
  • Reverse Staining Using Imidazole-Zinc

• PVDF Membranes
• Colloidal Gold Total Protein Stain
• Rapid Fluorescent Staining
• RUBY BLOT Staining
• Amido Black Staining
• Ponceau Staining

• Electroblotting
  • ABI Protocols - 4/11/05

* Note: All samples are shown using Transferrin (78kDa) and Carbonic Anyhydrase (29kDa). Markers used are SeeBlue Plus 2 Pre Stained Standard (Invitrogen) in Lane 1 and Mark 12 Wide Range Protein Standard (Invitrogen) in Lane 10.

Silver Staining

Sample Gel - Low Load 5pmol-0.025pmol*

Warning! Do not use silver stain methods that use glutaraldehyde. The glutaraldehyde prevents us from recovering any of the sample.

1. After electrophoresis, fix the gel slab in 50% methanol, 5% acetic acid in water for 20 minutes.
2. Then wash the gel slab for 10 minutes with 50% methanol in water and an additional 10 minutes with water to remove the remaining acid.
3. Sensitize the gel by a one-minute incubation in 0.02% sodium thiosulfate, and rinse it with 2 changes of distilled water for 1 minute each. (Developed gels are completely transparent when the sensitization step with sodium thiosulfate is included.)
4. After rinsing, submerge the gel in chilled 0.1% silver nitrate solution and incubate for 20 minutes at 4^oC.
5. After incubation, discard the silver nitrate and rinse the gel slab twice with water for 1 minute and then develop it in 0.04% formalin (35% formaldehyde in water) in 2% sodium carbonate with intensive shaking. After the developer turns yellow, discard it and replace it with a fresh portion. It is essential that the developing is carried out in an absolutely transparent solution.
6. After the desired intensity of staining is achieved, terminate the development by discarding the reagent, and then wash the gel slab with 5% acetic acid. The complete staining procedure should last no longer than 1 hour.
7. Store the silver-stained gel in a solution of 1% acetic acid at 4^oC until analyzed.

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RUBY GEL Stain (Biorad SYPRO RUBY GEL Stain # 170-3125)

Sample Gel - Low Load - 5pmol-0.025pmol*

1. Remove the gel from the gel cassette or plates. Place it in a plastic tray. Wash for 30 minutes in 10% methanol, 7% acetic acid prior to staining.
2. Remove the wash solution and cover the gel with RUBY GEL Stain (approx. 10 times the volume of the gel). Using too little stain will reduce the sensitivity.
3. Stain the gel with continuous agitiation for 3 hrs. for maximal sensitivity. Specific staining can be seen in 30-90 minutes. For convenience, gels may be left in the stain solution overnight (16-18 hours) without overstaining.
4. Rinse the gel in 10% methanol, 7% acetic acid for 30-60 minutes. This rinse step decreases background fluorescence.

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Pierce Gel Code Blue Staining Process (Pierce Product No: 24590)

Sample Gel - Low Load - 5pmol-0.025pmol*

1. Prewashing: After electrophoresis, transfer gel to a clean tray and rinse 3 times, 5 minutes each, with 100-200 ml of deionized water. Alternatively, wash gel in 1-2 L of deionized water with gentle shaking for 15 minutes.

2. Staining: Add GelCode Blue Stain Reagent to the tray. The amount of staining reagent required for the gel will depend on the size of the tray used. Typically, 20 ml of reagent will be sufficient for a 8 x 10 cm mini gel. Gently shake the tray and periodically check the protein band development. Staining intensity generally reaches a maximum within 1 hour. Staining overnight will not increase the background.

Note: PhastGel gels may require longer staining times (2 hours - overnight) for optimal band visualization.

Note: GelCode Blue Stain fixes most standard PAGE gels during the staining process. Additional fixing step(s) are usually not required.

3. Water Wash Enhancement Step: Replace the staining reagent with deionized water. (Several changes with deionized water may be necessary for optimal results.) This water equilibration enhances staining sensitivity as weak protein bands further develop in deionized water over a period of 1-2 hours.

Native PAGE Gel and Western Transferred Gel Staining

1. Rinse gel with deionized water for 5 minutes. Stain gel and perform Water Wash Enhancement Step as indicated in SDS-PAGE Gel Staining protocol above (Steps 2-3).

Western Transferred Proteins-PVDF Membrane Staining

1. Rinse PVDF membrane containing transferred proteins for 1-2 minutes with deionized water. Stain membrane and perform Water Wash Enhancement Step as indicated in SDS-PAGE Gel Staining protocol above (Steps 2-3).

Aternate Microwave Protocol

Prewashing:
1. After electrophoresis, transfer the gel to a microwavable tray containing 100 mL of deionized water and microwave for 90 seconds.
2. Discard water, replace with 100 mL of fresh deionized water, and microwave again for 90 seconds.
3. Replace water with fresh deionized water and place on an orbital shaker for 5 minutes.

Staining:
4. Discard the water and add 50 mL of GelCode Blue Stain Reagent. Microwave for 1 minute or until the solution begins to boil (make sure the gel is completely covered with stain reagent).
5. Place the tray on an orbital shaker and incubate for an additional 5 minutes.

Water Wash Enhancement Step:
6. Discard the staining reagent and replace with 200 ml of deionized water. Incubate for 10 minutes on an orbital shaker.

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Coomassie Staining

Sample Gel - High Load 100pmol-5pmol*

There are many methods of staining with Coomassie, all of which will work. The following is the way it is done in this laboratory.

Staining solution (500 ml):

• 250 ml MeOH
• 35 ml Glacial Acetic Acid
• 0.25 g Coomassie Blue R250
• add distilled H₂0 to 500 ml *Filter the solution before use.

  Destaining Solution (1000 ml)

• 100 ml MeOH
• 150 ml Glacial Acetic Acid
• add distilled H₂O to 1 liter

To stain gel, soak it in stain solution 3 hours to overnight. Remove staining solution and cover gel with destaining solution. Change destaining solution several times (every 30 minutes or so) until bands are visible.

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Colloidal Gold Total Protein Stain (BIO-RAD Colloidal Gold Total Protein Stain #170-6527)

Sample Blot - Low Load 5pmol-.025pmol*

TTBS Solution
Tween 20, Tris-Buffered Saline, 1XTTBS, 2L
20mM Tris
500mM NaCl
0.3% Tween-20, pH 7.5

Protocol

1. After blotting, transfer the membrane to an incubation vessel and wash (3 times) with TTBS solution (100mL each time, 20 minutes).
2. Rinse membrace (3 times) with dd water (2 minutes each) *water rinse is critical to remove all salts
3. Add Colloidal Gold Total Protein Stain (50mL) *all bands should be visible in 1-2 hours
4. Remove the gold stain solution and rinse (3 times) membrane with dd water (100mL, 1 minute each) *the gold solution can be reused until the gold is depleted - evident by the loss of the dark burgundy color and longer staining times.

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Reverse Staining Using Imidazole-Zinc

1. Rinse gel in dH₂O for 5 seconds after SDS-PAGE.
2. Soak gel in 0.2 M Imidazole with gentle agitation for 5-30 minutes depending upon gel concentration.
3. Immerse gel in 0.3 M zinc chloride (or zinc sulfate or zinc acetate) shaking gently for 1 minute or until background becomes opaque white with transparent bands.
4. Store in dH₂O or in sealed bags.

To destain simply wash the gel for 10 minutes with 2-3 changes in 2% citric acid.

One might try blotting after destaining the gel but it has not been done in this lab.

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RUBY BLOT Stain - PVDF (Biorad SYPRO RUBY BLOT Stain # 170-3127)

1. After electroblotting proteins to a PVDF membrane, allow membrane to dry completely.
2. Float the membrane face down in 7% acetic acid, 10% methanol and incubate for 15 minutes.
3. Wash the membrane in deionized water 4 times for 5 minutes each.
4. Transfer the blot using forceps to a staining dish containing RUBY BLOT Stain.
5. Float the membrane in RUBY BLOT Stain for 15 minutes.
6. Wash the membrane in deionized water 2 to 3 times for 1 minute each to remove excess stain. The membrane may be monitored periodically using UV epi-illumination to determine the level of background fluorescence.
7. Membranes stained with RUBY BLOT Stain are best preserved by allowing membranes to air dry. After air drying they can be handled without using forceps.

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Rapid Fluorescent Staining Procedure - Electroblotted PVDF Membranes

Sample Blot - Low Load - 5pmol-0.025pmol*

Sample Blot - Low Load - 5pmol-0.025pmol - flurorescent light*

1. After electroblotting the membrane, wash with deionized water to remove buffer salts (at least 2 changes of 100 ml, mild agitation over 15-20 minutes).
2. Blot membranes dry with filter paper, then dry in vacuo for at least 20 minutes.
3. Immerse dried membrane into a solution of 30% aq. methanol containing 0.2% acetic acid and 0.005% of sulforhodamine B for approx. 60 seconds with mild agitation.
4. Wash membrane with deionized water to remove excess dye.

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Amido Black Staining

Sample Blot - High Load 100pmol-5pmol*

Make a solution of 0.1% amido black in HPLC grade water. Filter. This solution can be stored at room temperature for several months.

1. Cover the membrane with stain.
2. Incubate approximately three minutes.
3. Other protocols say to only incubate 30-60 seconds, but I find this to be too short a time and I don't see any bands.
4. Destain with fresh HPLC grade water, changing several times.
5. Let membrane dry on clean blotting paper.
6. Store the membrane dry in the refrigerator or freezer. I keep them in ziploc bags or plastic wrap so they stay clean.

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Ponceau Staining Procedure (Sigma prod. number P7170)

Sample Blot - High Load - 100pmol-5pmol*

Total Protein Detection:

1. After transferring proteins from SDS-PAGE onto membrane, immerse the membrane in sufficient Ponceau Staining Solution and stain for 5 minutes.
2. Rinse membrane with deionized water until the membrane is clear.
3. Dry membrane under a heating fan.

Reversible Protein Detection:

1. After proteins are transferred onto membrane, immerse the membrane in Ponceau Staining solution and stain for 5 minutes.
2. After proteins have been visualized, rinse the membrane with distilled water and rapidly immerse in an aqueous solution of 0.1 M NaOH. Protein bands will start to disappear after 10-30 seconds.
3. Rinse membrane with running distilled water for 2-3 minutes.

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Electroblotting

1. All containers should be washed with acid (1 M HCl) and well rinsed with fresh HPLC grade water and then rinsed with HPLC grade methanol. Blotting sponges and apparatus should be treated this way as well.
2. Cut filter paper and PVDF membranes to size. Soak paper in transfer buffer.
3. Soak PVDF membranes in methanol to wet and then soak in transfer buffer.
4. Soak blotting sponges in transfer buffer.
5. After electrophoresis rinse the gel briefly in transfer buffer to remove residual glycine from electrophoresis buffer.
6. Assemble blotting sandwich as follows: from positive to negative electrodes (black to red)
   1) blotting sponges (1 or 2 as needed),
   2) filter paper,
   3) gel,
   4) PVDF membrane,
   5) filter paper,
   6) blotting sponge.
   Repeat from steps 2 to 6 to transfer a second gel.
7. Blot at 25 V for 1.5 hours. This is effective for proteins 65 kD and below. Longer times or slightly higher voltages are necessary for larger proteins.
8. After the blotting procedure is finished, stain the membrane to visualize the protein bands.