Salmaso Lab TRAP Adaptation

Stephanie Simard

Mar 28, 2025

Salmaso Lab TRAP Adaptation

Forked from a private protocol

DOI

dx.doi.org/10.17504/protocols.io.n2bvj311wlk5/v1

Stephanie Simard¹

¹Carleton University

Stephanie Simard

Carleton University

DOI: dx.doi.org/10.17504/protocols.io.n2bvj311wlk5/v1

Protocol Citation: Stephanie Simard 2025. Salmaso Lab TRAP Adaptation. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj311wlk5/v1

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this protocol and it's working

Created: January 09, 2024

Last Modified: March 28, 2025

Protocol Integer ID: 93163

Keywords: ASAPCRN

Abstract

This protocol details the procedure of Salmaso Lab TRAP Adaptation.

Attachments

516-1071.docx

34KB

Guidelines

Note
The following protocol is for 3-day TRAP; 3-Day protocol allows maximal coating of magnetic beads with anti-eGFP.


The maximum number of IP that may be performed using a single 1.5 mL tube is limited to 1mL of reagent; this works out to a MAXIMUM of 6 IPs per tube, this protocol should be limited to using a maximum of 20 IPs per 3-Day protocol (limits of the equipment).


The main goals of the protocol are as follows:

Preparing the magnetic beads for incubation with your sample. This is achieved through washing the beads and then coating them in Protein L and then incubating the coated beads with anti-GFP
Homogenizing your tissues and preparing them to be incubated with anti-GFP coated magnetic beads
Extracting the mRNA that has been extracted from your tissues currently bound to the anti-GFP magnetic beads
Use RNAse Zap wipes throughout (Ambion AM#9786)

Materials

Day 1: Coating the Magnetic Beads & Preparing Stock Solutions

Solutions:

ReagentInvitrogen Dynabeads™ MyOne™ Streptavidin T1Thermo Fisher ScientificCatalog #65601  
ReagentPierce&trade; Recombinant Protein L, BiotinylatedThermo FisherCatalog #29997  
Anti-GFP antibodies (made for TRAP; stored at Temperature-20 °C  )

Stock Concentration0.15 Molarity (M)   & 0Concentration35 Molarity (M)   KCl Wash Buffer, Homogenization buffer (for Day 2)

 Reagent1M MgCl2AmbionCatalog #AM9530G  
 Reagent2M KClInvitrogen - Thermo FisherCatalog #AM9640G  
 ReagentSurfact-Amps NP-40Thermo Fisher ScientificCatalog #28324  
    1M DTT  (added immediately before use) 
    CHX-MeOH (added immediately before use)

ReagentPBS - Phosphate-Buffered Saline (10X) pH 7.4Thermo Fisher ScientificCatalog #AM9625  
Protease-free BSA (make sure it is IgG free; BioShop#9048-46-8)




Materials & Equipment:

Reagent1.5 ml micro-centrifuge tubeAmbionCatalog #AM12450  
Magnetic stand with tube carriage
Slow-rotating rotisserie 
Access to a walk-in cold room or a cold fridge with electrical outlets 


Stock Solution Preparation

Prepare the stock solutions of Protein L, Concentration0.15 Molarity (M)   KCl Wash Buffer, Concentration0.35 Molarity (M)   KCl Wash Buffer, & Homogenization Buffer using sterile, biochemical grade water to save time during subsequent days.

Reconstitute Biotynlyated Protein L to Concentration1 µg/µL   with sterile, TRAP-grade 1x PBS. Be sure to wash the sides of the bottle with the 1x PBS and swirl gently to dissolve the Protein L.


To prepare a stock 100 mL of 0.15M KCl Wash Buffer:

Gather a 100mL biochemical grade sterile water container. Remove approximately 25mL into a CLEAN beaker, set aside for later use.

To the biochemical water container add:

       a.Amount1 mL   of HEPES-KOH buffer
       b.Amount500 µL   of MgCl2
       c.Amount7.5 mL   of KCl
       d.Amount10 mL   of 10% NP-40 (one glass vial; use caution when opening)

Using the water set aside, fill container back to Amount100 mL   and mix manually.
Note
This is not the final solution; immediately before use you will need to add activated CHX & DTT.


To prepare a stock 100mL of 0.35M KCl Wash Buffer

Gather a 100mL biochemical grade sterile water container. Remove approximately Amount30 mL   into a CLEAN beaker, set aside for later use.

To the biochemical water container add:

      a.Amount1 mL   of HEPES-KOH buffer
      b.Amount500 µL   of MgCl2
      c.Amount17.25 mL   of KCl
     d.Amount10 mL   of 10% NP-40 (one glass vial; use caution when opening)

Using the water set aside, fill container back to Amount100 mL   and mix manually.
Note
This is not the final solution; immediately before use you will need to add activated CHX & DTT.


To prepare a stock 100mL of Homogenization Buffer
Note
You will need Amount1 mL   of buffer PER sample (n); it is best to make more than required for the entire experiment to avoid having the remake more before a run.


 
Gather a 100mL biochemical grade water container. Remove approximately Amount10 mL   into a CLEAN beaker, set aside for later use.

To the biochemical water container add:

              a.Amount1 mL   of HEPES-KOH
              b.Amount500 µL   of MgCL2
              c.Amount7.5 mL   of KCl

Using the water set aside, fill container back to Amount100 mL   and mix manually.
Note
This is not the final solution; immediately before use you will need to add activated CHX, DTT, Superasin, Protease Inhibitor Tablet.


 
Day 2: Tissue Homogenization & Bead Incubation

Materials Needed:

Previously prepared stock solutions: 
         1. Homogenization buffer
          a. Protease inhibitor tablets, SUPERase, CHX, DTT
        2. Concentration0.15 Molarity (M)    KCl Wash Buffer
         a. CHX, DTT
10% NP-40 (in a glass vial)
ReagentDHPCAvanti Polar Lipids, Inc.Catalog #850306  
Large tubes (5mL), flat bottom preferably 
New set of 1.5mL low-bind tubes 
PCR tubes


Day 3: mRNA Extraction & Quantification

Materials Needed:

ReagentAbsolutely RNA Nanoprep Kit 50 prepsAgilent TechnologiesCatalog #400753  
     1.Lysis Buffer
     2. βME
     3. DNAse 
    4.Salt Washes
Concentration0.35 Molarity (M)   KCl Wash Buffer
80% Sulfolane (warmed on the dry bath)

Before start

Day 3: mRNA Extraction & Quantification:

Before beginning; turn on the incubator to Temperature37 °C  ; and the dry bath to Level 3-5 on the “low” setting. Place the sulfolane bottle on top of the dry bath to warm and liquify. 

Day 1: Magnetic Bead Preparation

Note
The below information is for 6 IPs, with single values in parentheses.
It is important to note that each run should encompass at least 1 whole N (one IP from each group) from your experiment.


Gather the magnetic beads; pipet the solution up and down to mix the beads.
Note
The magnetic beads settle on the bottom of the bottle and need to be mixed gently to ensure proper distribution and concentration.

Extract Amount360 µL   of the magnetic beads into a 1.5mL low-bind tube [Amount60 µL   per IP]. Place the tube into the tube carriage and place onto the magnet. Wait for the beads to settle on the walls of the tube.

Wash beads 3 times: Discard the excess liquid from the tube, wash with Amount1 mL   of 1x PBS. Remove the tube carriage and shake to suspend magnetic beads in 1x PBS. Replace the magnet, discard the excess 1x PBS and repeat 2 more times. 

Collect the beads on the magnet and remove all excess liquid. Incubate the beads with Amount144 µL   of Protein L (Concentration1 µg/µL  ) [Amount24 µL   per IP]. Fill the tube with 1x PBS for a total volume of Amount1 mL  , in this case Amount496 µL   of 1x PBS. 

                (Amount360 µL   beads + Amount144 µL   Protein L + Amount496 µL   1 x PBS = Amount1000 µL  , Amount1 mL  )

Place the tube in the rotating rotisserie and spin slowly for ~Duration01:00:00   at TemperatureRoom temperature  .
Note
Incubating beads with Protein L allows the beads to become coated in protein that is required to bind anti-eGFP.

While beads rotate, prepare Amount10 mL   of 3% BSA in 1x PBS (weight/volume).

Measure out Amount300 mg   of solid BSA and add to a 15mL conical tube.

Top up conical tube to Amount10 mL   with 1x PBS, mix vigorously until BSA is dissolved.

Also prepare Amount10 mL   of FINAL Concentration0.15 Molarity (M)   KCl Wash Buffer in a 15mL conical tube by adding:

Amount5 µL   Concentration1 Molarity (M)   DTT.
Safety information
Very toxic, dispose of in hazardous wastes; pungent smell.

Amount10 µL   activated 1000x CHX (100% MeOH, see dissection protocol).

After beads have been incubated with Protein L, collect the beads using the magnet and discard excess liquid. Wash the beads with the 3% BSA solution 5 times.

Note
Washing with high volume of BSA collects the excess Protein L that has not been bound well to the magnetic beads.

After the final wash, resuspend the beads with Amount175 µL   of Concentration0.15 Molarity (M)   KCl and Amount47 µL   of anti-GFP solution. Rotate DurationOvernight   (~Duration24:00:00  ) in a walk-in cold room, or fridge (Temperature4 °C  ).

Day 2: Tissue Homogenization and Bead Incubation

Note
You will need a large bin of ice to keep samples cold between uses, for the beginning steps leave beads rotating in the cold room until samples are ready to be homogenized. Day 2 incubates the magnetic beads with sample to extract mRNA. 

Note
Try to keep samples TemperatureOn ice   whenever possible; do not let them sit at TemperatureRoom temperature   for too long. Be sure to clean all surfaces being used with RNAse wipes. Day 2 requires utmost care for being clean and sterile. 

Prepare 1mL/sample of homogenization buffer from stock solution. Measure into a 15mL conical tube (try to make at least Amount10 mL   at a time; up to Amount12 mL   at a time maximum).

Add 1 protease inhibitor tablet per Amount10 mL   (can use 1 tablet up to Amount12 mL  ) (Fisher #A32955).

Amount1 µL   CHX per Amount1 mL   of homogenization buffer.

100units SUPERase (RNAse inhibitor) per sample (Ambion #AM2694).

Amount5 µL   DTT per  Amount10 mL   of homogenization buffer (Concentration0.5 Mass Percent  ).

Mix the solution well, vigorously and until the protease inhibitor has dissolved completely (this may take a few minutes). Set aside TemperatureOn ice  , keep cold.

Retrieve samples from freezer. Move sample to a new, labelled 5mL tube TemperatureOn ice  . Add Amount1 mL   of homogenization buffer to each sample. Let sit TemperatureOn ice   (to thaw) for ~ Duration00:05:00   .

Note
 If you are doing fresh dissections, you do not need to let samples thaw TemperatureOn ice  .

In a FUME HOOD, set up homogenization blade or pestle. Gather enough dH2O in a beaker to be able submerge the end of the blade or pestle. Turn on blade to a slow setting and submerge into homogenization buffer, moving the sample up and down until completely homogenized. Sample will be yellow and completely liquid. Clean blade with RNAse wipe + dH2O between each sample.

Transfer homogenized tissues back into a new, labelled 1.5mL low-bind tube. Spin at Centrifigation20000 x g  for Duration00:10:00   at Temperature4 °C  . Collect supernatant, do not disturb the pellet, into a new, labelled 1.5mL low-bind tube.

10m

Add Amount100 µL   10% NP-40 & Amount115 µL   DHPC (use a 1ml syringe because it is very thick/viscous) to each sample, mix with inversion and let sit TemperatureOn ice   for Duration00:05:00  .

Spin the samples at Centrifigation20000 x g  for Duration00:15:00   at Temperature4 °C  .

15m

During the 15-minute cycle, begin to prepare the beads to be incubated with the sample. Prepare ~Amount10 mL  - Amount15 mL   of Concentration0.15 Molarity (M)   KCl Wash Buffer.

Suspend the beads on the magnet, discard excess liquid and wash 3 times with Amount1 mL   of Concentration0.15 Molarity (M)   KCl wash buffer.

Note
Remember that these wastes contain CHX, DTT and need to be discarded in chemical waste.

After the 3rd wash, resuspend the beads using Amount1080 µL   Concentration0.15 Molarity (M)   KCl Wash Buffer + Amount120 µL   DHPC [Amount180 µL   Concentration0.15 Molarity (M)   KCl, Amount20 µL   DHPC per sample].
Note
Following 15-minute cycle; collect supernatant.

Collect post-mitochondrial (S20), take care to avoid disturbing the pellet, into a new 1.5 tube. Collect Amount50 µL   of the S20 post-mitochondrial supernatant into a new PCR tube, label and freeze at  Temperature-80 °C  . 
Note
This S20 will act as “input” for validation of the model.

Add Amount200 µL   of the bead solution to each sample, place on the rotisserie and let rotate DurationOvernight   at Temperature4 °C  .

10m

Day 3: mRNA Extraction and Quantification

42m

Note
It is vital to take care and use RNAse wipes liberally to keep surfaces and materials clean. Day 3 requires utmost care to keep samples cold to prevent RNA degradation. First you will need to strip the beads; then you will be able to extract the mRNA.


Prepare Amount20 mL   of FINAL Concentration0.35 Molarity (M)   KCl  in a 50mL conical tube by adding:

Amount10 µL   Concentration1 Molarity (M)   DTT.
Safety information
Very toxic, dispose of in hazardous wastes; pungent smell.

Amount20 µL   activated 1000x CHX (in 100% MeOH, see dissection protocol).

Retrieve the rotating samples from the rotisserie and collect in the magnet caddie, wait until beads have settled on the magnet. Extract the liquid into a labelled 1.5mL tube, this is the “unbound” fragment used for validation.
Note
The beads here contain the mRNA that you are interested in, be careful to collect all the beads before collecting the liquid portion.

Wash the beads with Amount200 µL   of  Concentration0.35 Molarity (M)   KCl  Wash Buffer 4 times.

During the 4th wash, prepare the lysis solution in the fume hood by mixing Amount600 µL   lysis buffer with Amount4.2 µL   βME  in a 1.5mL tube. 
Note
Both can be found in the extraction kit.

(Amount100 µL   Lysis buffer, Amount0.7 µL   βME per sample)

Resuspend each of the sample beads with Amount100 µL   of the prepared lysis solution, mix well and let incubate at TemperatureRoom temperature   for Duration00:10:00  . Take this time to label out a new set of tubes, as well as the filters from the RNA extraction kit.
Note
Be careful when handling the filter cup not to disturb the filter, wash any surface that the tubes or your hands will touch with RNAse wipes.

10m

Place the samples back on the magnet and collect the liquid (which now has the mRNA) into a new, labelled, low-bind 1.5mL tube; begin with RNA extraction from the solution.

Add Amount100 µL   of the warmed 80% sulfolane to each of the samples and mix by inversion. 
Note
The sulfolane should be warm, not hot.

Make the DNAse soltution by combining Amount15 µL    reconstituted DNAse with Amount75 µL    DNAse Digestion Buffer. Set aside for now.

(Amount2.5 µL   DNAse, Amount12.5 µL   DNAse Digestion Buffer per sample)

Put the whole sample into a labelled filter cup with a collection tube underneath, cap and spin at Centrifigation12000 x g  for Duration00:01:00   at Temperature4 °C  .
Note
The filter cup now contains your RNA, keep track of the filters.

Discard the liquid in the collection tube (filtrate), add Amount300 µL   Low-Salt Wash  to the filter cup, spin at Centrifigation12000 rpm  for Duration00:01:00   at Temperature4 °C  . Discard the filtrate and spin at Centrifigation12000 x g  for Duration00:02:00   to dry the filter.

Add Amount14.5 µL   DNAse + Digestion Buffer directly onto the filter, taking care not to touch the filter of each sample, incubate at Temperature37 °C   for Duration00:15:00  . 
Note
During this time, gather Amount40 µL   Elution Buffer (from kit) per sample in a 1.5mL tube, and heat in the warm bath until needed, collect and label PCR tubes to collect RNA for the run, use a cold caddy to keep the tubes cold. 

15m

Add Amount300 µL   High-Salt Wash to each sample, spin at Centrifigation12000 x g  for Duration00:01:00   at Temperature4 °C  . Discard filtrate.

Add Amount300 µL    Low-Salt Wash to each sample, spin at Centrifigation12000 x g  for Duration00:01:00   at Temperature4 °C  . Discard filtrate.

Add Amount300 µL    Low-Salt Wash to each sample, spin at Centrifigation12000 x g  forDuration00:03:00   at Temperature4 °C  . Discard filtrate.

Spin at Centrifigation12000 x g  for Duration00:01:00   at Temperature4 °C  . Discard filtrate and collection tube. Replace with a new collection tube.

Add Amount20 µL   heated Elution Buffer directly to the filter of each sample, incubate at  TemperatureRoom temperature   for Duration00:02:00  .

Spin samples at Centrifigation12000 x g  for Duration00:05:00   at Temperature4 °C  .

Collect the resulting filtrate into a cold PCR tube, label as the “Primary Elution” (1°E).

Repeat steps 34 & 35, labelling the second set of PCR tubes as “Secondary Elution” (2°E).

Bring 1°E & 2°E to the NanoDrop; record the RNA concentrations for each sample and the 260nm/280nm values.

Freeze the samples at Temperature-20 °C   for short term use (~2 weeks) or Temperature-80 °C   for a longer period (good for up to 2 years, depending on storage conditions).

Public workspaceSalmaso Lab TRAP Adaptation

Salmaso Lab TRAP Adaptation