Immunoprecipitation

Agnes Roczniak-Ferguson; Shawn M. Ferguson

Jun 06, 2024

Immunoprecipitation

DOI

dx.doi.org/10.17504/protocols.io.5jyl822z7l2w/v1

Agnes Roczniak-Ferguson^1,2,3,4,5,
Shawn M. Ferguson^1,2,3,4,6,5

¹Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
²Department of Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
³Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
⁴Wu Tsai Institute, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
⁵Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA.;
⁶Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA

Berrak Ugur

Yale University

DOI: dx.doi.org/10.17504/protocols.io.5jyl822z7l2w/v1

Protocol Citation: Agnes Roczniak-Ferguson, Shawn M. Ferguson 2024. Immunoprecipitation. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl822z7l2w/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: May 24, 2024

Last Modified: June 06, 2024

Protocol Integer ID: 101276

Keywords: ASAPCRN

Funders Acknowledgements:

ASAP

Grant ID: 000580

Abstract

This protocol details the immunoprecipitation of Hela cells.

Materials

1% Triton X100 lysis buffer
AB
1% Triton X-100 Buffer:Amount/Liter
Tris, 50 mM6.057 g
NaCl, 150 mM	8.766 g
Triton X-100, 1%10.0 ml
Pierce Bradford Assay reagent
ABCD
Coomassie Reagent (ml)BSA (2 µg/µl) volume (µl)BSA concentration (µg/µl)Sample (µl)
1 0 0 0
1 1 2 0
1 2 4 0
1 4 8 0
1 8 160
1 2
ReagentPierce&trade; Coomassie (Bradford) Protein Assay KitThermo FisherCatalog #23200 

10 x Sample buffer
AB
Chemical Amount/20 ml
0.5 M Tris pH 6.810 ml
10% SDS2 g
50% Sucrose10 g
0.04% Bromophenol Blue8 mg
1.2 M Mercaptoethanol2 ml

Seed Hela cells into 150 mm dishes at a density of 1.9 million cells.  The cells will be near confluent in 2 days.  Otherwise seeding 4x106 of cells/150 mm dish will be near confluent the next day.  Expect to get 3-Amount5 mg   of protein per dish.  These numbers will be different for other cell lines and will have to be determined prior to the experiment. 

Turn on the centrifuge, install rotor for 1.5 ml tubes and chill to Temperature4 °C  .

To 1% Triton X100 lysis buffer add Roche Complete Mini, EDTA-free protease inhibitor (1 tablet/10 ml) and Roche PhosSTOP phosphatase inhibitor (1 tablet/10 ml).  Allow to dissolve, vortexing occasionally. 
AB
1% Triton X-100 Buffer:Amount/Liter
Tris, 50 mM6.057 g
NaCl, 150 mM8.766 g
Triton X-100, 1%10.0 ml
Adjust pH to Ph7.4 

Prepare ice, sample tubes, PBS for washing cells, cell scrapers and a beaker for decanting media.

Decant media into a beaker and wash cells 3x with Amount20 mL   of ice-cold PBS/150 mm dish, each time decanting into the beaker.  After the last wash aspirate off as much media as possible.

Place cell culture dish TemperatureOn ice  , add Amount1 mL  /150 mm dish of 1% Triton X100 + dissolved protease inhibitors, scrape the cells off using a cell scraper and transfer the cell mixture into a chilled microcentrifuge tube.  If using several dishes, harvest one dish and then transfer the lysate to the next dishes to minimize the volume.

Spin lysates for Centrifigation20000 x g, 4°C, 00:05:00 . Collect the supernatant and place in new chilled microcentrifuge tubes.

Prepare standards and samples as indicated in the table for analysis of protein concentrations using the Pierce Bradford Assay reagent (ThermoFisher Scientific catalog # 23200).

ABCD
Coomassie Reagent (ml)BSA (2 µg/µl) volume (µl)BSA concentration (µg/µl)Sample (µl)
1 0 0 0
1 1 2 0
1 2 4 0
1 4 8 0
1 8 160
1 2

Measure protein concentration using the NanoDrop or other spectrophotometer.

For immunoprecipitation, prepare the Chromotek GFP-TRAP agarose resin by aliquoting Amount10 µL   of bead suspension per sample. Make sure that the beads are well resuspended before pipetting. To facilitate pipetting cut off the ends of the micropipette tips. Amount20 µL   of unconjugated agarose beads can be added per sample to help with visualizing the bead pellet.

Wash beads 3x with Amount1 mL   lysis buffer.  For each wash invert tube up and down a few times, pellet the resin by centrifugation for Centrifigation10.000 x g, 00:00:30  and carefully aspirate the supernatant.

30s

After the last wash resuspend the resin in a small volume of lysis buffer and divide it equally between sample tubes.  Spin again and aspirate off excess lysis buffer.

Transfer cell lysates into the tubes containing resin (use equal protein amounts for each sample typically ~ Amount1 mL   of 3-Amount5 µL   lysate).  Save 10% of total cell lysate to use as a control during immunoblotting.

Rotate immunoprecipitation samples end-over-end at Temperature4 °C   for Duration01:00:00  .

At the end of the incubation centrifuge Centrifigation10000 x g, 00:00:30  to collect beads; aspirate most of supernatant using a vacuum and super fine tip, being careful not to disrupt the pellet.

30s

Wash pelleted beads 4-6x with lysis buffer (Amount1 mL   for each wash).  For each wash, mix the suspension for ~Duration00:00:03  , spin down (~Duration00:00:10   pulse), and aspirate supernatant. To be safe, do not try to get all of it -- leave some (5%) buffer over the beads. Work quickly and keep samples cold.

13s

Using a fine pipette tip, remove remaining supernatant from the beads after last wash.  Place tubes back TemperatureOn ice  .

Add Amount50 µL   of 2X sample buffer to each tube of beads; vortex at ~1/2 speed, keeping beads in bottom of tube.

10 x Sample buffer
AB
Chemical Amount/20 ml
0.5 M Tris pH 6.810 ml
10% SDS2 g
50% Sucrose10 g
0.04% Bromophenol Blue8 mg
1.2 M Mercaptoethanol2 ml

Denature samples at Temperature95 °C   for Duration00:03:00  . 

Centrifuge at Centrifigation10000 x g, 00:00:30 .

30s

Transfer supernatant to new tube using a fine pipette tip. (You can pick up residual sample from the beads by pressing the fine pipette tip against bottom of tube and rocking it back and forth, excluding beads).

Samples may be frozen at this point, or you can proceed to immunoblotting.

	A	B
	1% Triton X-100 Buffer:	Amount/Liter
	Tris, 50 mM	6.057 g
	NaCl, 150 mM	8.766 g
	Triton X-100, 1%	10.0 ml

A	B	C	D
Coomassie Reagent (ml)	BSA (2 µg/µl) volume (µl)	BSA concentration (µg/µl)	Sample (µl)
1	0	0	0
1	1	2	0
1	2	4	0
1	4	8	0
1	8	16	0
1			2

	A	B
	Chemical	Amount/20 ml
	0.5 M Tris pH 6.8	10 ml
	10% SDS	2 g
	50% Sucrose	10 g
	0.04% Bromophenol Blue	8 mg
	1.2 M Mercaptoethanol	2 ml

Public workspaceImmunoprecipitation

Immunoprecipitation