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sds_dtt_protein_extraction_from_ffpe_material.md

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protocol

  • Protocol: SDS/DTT Protein Extraction From FFPE Material
  • Faculty: Cumming School of Medicine
  • Institute: Charbonneau Cancer Institute
  • Site: Arthur J.E. Child Comprehensive Cancer Center, YC
  • Version: 2
  • Version Date: 5SEPT2024
  • Approver: ermahe@ucalgary.ca

authorization

  • Requires completion of site specific safety training
  • Requires pre-authorization by PI

hazards

  • Xylene, DTT and ethanol are toxic, volatile, flammable, and/or poisonous.
  • SDS is a potent solvent
  • Scrape-blades/glass slides may cause sharps injury
  • Thermomixer may cause thermal injury

ppe

  • Lab Coat
  • Eye Protection
  • Nitrile Gloves

where

  • Fumehood
  • Pre-PCR bench

references

  • NA

emergency

  • If skin/eye contact with chemicals, flush aggressively and continuously with water using an eyewash, sink or shower
  • Use spill kit to clean up small spills; Contact maintenance and log incident for large spills
  • Contact emergency services and log any injuries

cleanup

  • Benchtop/fumehood surface wipedown with 70% isopropanol

waste

  • Xylene/ethanol should be disposed of in chemical waste container (fume hood)
  • Dispose of sharps (blades and slides) in yellow biohazard tubs (NOT biohazard bags)
  • Dispose of tips/tubes in biohazard bags or tubs

maintenance

  • NA

before_starting

  • Ensure specimens are accessioned and that sufficient labelled tubes are available
  • Block out approximately 3 hours and 30 minutes
  • If required, make 1M Tris stock: Dissolve 121 g Tris base in 800 ml ddH2O; warm to dissolve as required; adjust to desired pH with concentrated HCl (approximately 42 mL HCl is needed to achieve pH 8.0 solution); top up to 1L total volume with ddH2O
  • If required, make 10x SDS-Tris Stock: Dissolve 40 g SDS in ~80 mL of 1M Tris solution; warm to dissolve as required (place container in boiling water and mix agressively until clear, needs to be done every use of 10x SDS-Tris Stock); add additional 1M Tris up to 100 mL; store at 4°C
  • If required, make 1M DTT Stock: Slowly dissolve 1.55 g of DTT in 10 mL ddH2O, dispense into Eppendorf tubes in 1-mL aliquots, and store in the dark (eg wrapped in foil) at -20°C
  • Enter the accession numbers of the specimens in the table below to calculate the required volumes of reagents required

calculator

<!DOCTYPE html>
<html lang="en">
	<head>
		<meta name="viewport" content="width=device-width, initial-scale=1.0">
		<link rel="stylesheet" href="mahe-lab_style.css">
		<title>Working Solution Calculator</title>
	</head>
<body>
<!---configure reagent prep details here, with calculations based on the table inputs below--->
<table id="table0">
	<tr><td>Scan/enter the specimen accession numbers:</td><td></tr>
</table>
<table id="table1">
	<tr><td><input type="text" size="10px" id="00"/></td><td><input type="text" size="10px" id="01"/></td><td><input type="text" size="10px" id="02"/></td><td><input type="text" size="10px" id="03"/></td></tr>
	<tr><td><input type="text" size="10px" id="10"/></td><td><input type="text" size="10px" id="11"/></td><td><input type="text" size="10px" id="12"/></td><td><input type="text" size="10px" id="13"/></td></tr>
</table>
<br>
<button type="button" class="button" onclick="addRows()">Add Rows</button>
<br>
<!--- insert the necessary calculations here.--->
<table id="table2">
	<tr><td></td><td></td></tr>
	<tr><td>Working Solution Preparation</td><td></td></tr>
	<tr><td>Volume of required Working Solution Assuming ~10 mg of tissue per sample (uL):</td>
	<td><input readonly type="text" size="10px" id="TotalVol"/></td></tr>
<!--- calculation: --->
	<tr><td>Volume of 10x SDS-Tris Stock Required for Working Solution (uL):</td>
	<td><input readonly type="text" size="10px" id="calc1"/></td></tr>
<!--- calculation: --->
	<tr><td>Volume of 1M DTT Required for Working Solution (uL):</td>
	<td><input readonly type="text" size="10px" id="calc2"/></td></tr>
<!--- calculation: --->
	<tr><td>Volume of ddH2O Required for Working Solution (uL):</td>
	<td><input readonly type="text" size="10px" id="calc3"/></td></tr>
</table>
<br>
<button type="button" class="button" onclick="calculate()">Calculate</button>
<script type='text/javascript'>
function addRows() {
    var table = document.getElementById('table1');
    var trows = table.rows.length;
    var tcols = table.rows[0].cells.length;
    var row = table.insertRow(trows);
    for (var i=0;i<tcols;i++) {
	var txt = document.createElement('input')
	txt.setAttribute('type','text');
	txt.setAttribute('size','10px');
	txt.setAttribute('id',`${trows}${i}`);
	var col = row.insertCell(i);
	col.appendChild(txt);
    }
}	
function calculate() {
    var count = 0;
    var table = document.getElementById('table1');
    var trows = table.rows.length;
    var tcols = table.rows[0].cells.length;
    for (var i=0;i<trows;i++) {
        for (var j=0;j<tcols;j++) {
            var txtcontent = document.getElementById(`${i}${j}`).value;
            if(txtcontent != '') {
                count++;
            }
        }
    }
    //console.log(count)
    if(count>0) {
	document.getElementById('TotalVol').value = (110 * (count)).toFixed(1);
        document.getElementById('calc1').value = (11 * (count)).toFixed(1);
        document.getElementById('calc2').value = (11 * (count)).toFixed(1);
        document.getElementById('calc3').value = (88 * (count)).toFixed(1);
    }
}
</script>
</body>
</html>

procedures

  1. IF CURLS/SCROLLS ARE PROVIDED: Ensure the tube(s) are correctly labelled; move to step 5
  2. IF SLIDES ARE PROVIDED:
  3. If coverslipped, soak with xylene in a Coplin jar in fume hood for ~4 hours; use gentle traction (applied using forceps or another slide) to remove the coverslip
  4. Use fresh blade to scrape off the materials into a labelled 1.5 mL microfuge tube, try to remove the wax as one piece by curling it before sliding it into a tube. After each slide tap tube against desktop to move material to the bottem to mitigate static electricity.
  5. In the fumehood, add 1 ml xylene per labelled tube containing the FFPE tissue materials.
  6. Vortex 10 s and incubate 5 min at RT
  7. Centrifuge 3 min at 16,000 × g at RT and pipette off the supernatant
  8. In the fumehood, add 1 ml xylene per labelled tube containing the FFPE tissue materials.
  9. Vortex 10 s and incubate 5 min at RT
  10. Centrifuge 3 min at 16,000 × g at RT and pipette off the supernatant. Pellet can be transparent and difficult to see, tip tube sideways and press the pipette against the tube wall while pipetting off supernatent to avoid removing the pellet. Put supernatants in organic waste.
  11. In the fumehood, add 1 ml 100 % ethanol per tube
  12. Vortex 10 s and incubate 5 min at RT
  13. Centrifuge 3 min at 16,000 × g at RT and pipette off the supernatant
  14. In the fumehood, add 1 ml 95 % ethanol per tube.
  15. Vortex 10 s and incubate 5 min at RT
  16. Centrifuge 3 min at 16,000 × g at RT and pipette off the supernatant
  17. In the fumehood, add 1 ml 70 % ethanol per tube.
  18. Vortex 10 s and incubate 5 min at RT
  19. Centrifuge 3 min at 16,000 × g at RT and pipette off the supernatant
  20. Spin down, remove all remaining supernatant and let the tube stand open in the hood for 5 min to dry the pellet
  21. Add ~100 μL extraction buffer per ~10 mg tissue and gently pipette up & down to dissolve (Typical of 5 extracted slides)
  22. Incubate at 100 °C for 20 min at 500 rpm in the thermomixer
  23. Incubate at 60 °C for 2 h at 500 rpm in the thermomixer
  24. Centrifuge for 10 min at 16,000 × g, collect the extracted protein supernatant and transfer it to a clean labelled tube
  25. Proceed to Qubit protein quantitation; store extracts at −20°C