Bridges Lab Protocols - User contributions [en]

GST-EEA1 Pulldown from Yeast

2012-07-31T17:48:21Z

Sheelak: added link to 2xHNG

== '''Materials''' ==
*[[ 2xHNG_Buffer | 2x HNG ]] (100mL). Combine 100mM HEPES (10mL), 300mM NaCl (7.5mL), 20% glycerol (20mL)
*1x HNG (10mL). Mix 5mL 2xHNG and 1 PI tablet.
*Lysis Buffer (10mL). Combine 5mL 1x HNG, 1 PI tablet, 1mL of 1% NP40, and 50µL of magnesium chloride.
*Glutathione sepharose beads
*Glass beads

----

== '''Protocol''' ==
*Inoculate cells in appropriate media overnight.
*Re-suspend cells in 1mL of lysis buffer.
*Lyse cells: place approximately 1mL of glass beads into sonicator tube (Weisman Lab) and run in the sonicator 3x for 20 seconds. (Invert tube in between each round)
*Centrifuge at 4°C for 10 minutes.
*Add 1.5mL of 1x HNG to GST and GST-EEA1 proteins. (-80°C)
*Combine 450µL of protein with 450µL of lysates.
*Place tubes end over end for 30 minutes at 4°C.
*Add 50µL of glutathione sepharose beads to each tube.
*Wash each tube with 1x HNG five times at 4°C.
*Load in a 4-12% gel.

GST-GTPase Pull Down Assay

2012-07-31T17:46:53Z

Sheelak: wrote initial page

[[ Category: Protein Purification ]]
[[ Category: Protein-Protein Interactions ]]
[[ Category: Protein Biochemistry ]]
[[ Category: Proteins ]]
__NOTOC__

==Buffers==
Prepare each of these and chill on ice:
* Nucleotide Stripping Buffer: 5 mL [[ 2xHNG_Buffer | 2x HNG ]] + 5 mL water + 20 uL 0.5M EDTA
* Loading Buffer: 5 mL [[ 2xHNG_Buffer | 2x HNG ]] + 5 mL water + 100 uL 1M MgCl
* Lysis Buffer: 5 mL [[ 2xHNG_Buffer | 2x HNG ]] + 4.5 mL water + 500 uL 20% Triton X100 + PI Tablet
* Wash Buffer: 25 mL [[ 2xHNG_Buffer | 2x HNG ]] + 25 mL water

==Preparation of Immobilized GTPases==
#Make 20 ug aliquots of GTPases immobilized to glutathione beads (see [[ GST Pulldown Assay ]])
#Resuspend one aliquot in 1 mL Nucleotide Stripping Buffer
#Incubate at Room Temperature for ~ 20min
#Spin down beads, aspirate supernatant and wash 1 x with 1 mL Loading Buffer.
#Aspirate supernatant and add 500 uL Loading Buffer plus 10 uL GDP or GTPyS
#Incubate at room temperature for about 20 min

==Preparation of Lysates==
#Wash cells 2x with 25 mL PBS (for 150 mm plate; use 10 mL for 100 mm dish)
#Add 2 mL Lysis buffer (or 1 mL for 100 mm dish) and scrape cells using plastic spatula. Collect in 1.5 mL eppendorf tubes
#Incubate end over end for 20 min at 4 C
#Centrifuge 10 min at 13 000 RPM at 4C to clarify
#Remove supernatants to fresh tubes.
#Save a lysate sample in 5X SDS-Cocktail

==Pull-Down Assay==
#Add appropriate amount of lysate to nucleotide-loaded beads. Typically we use 2 ug for a pull-down, so that corresponds to 1/5 of the resuspended GST-GTPase solution. You can vary the beads and the lysate but a good starting point is 500 ug protein lysate. If necessary bring volume of assay up to ~1 mL with HNG
#Add 50 uL of Glutathione Beads for bulk during washing.
#Incubate end over end at 4C for 30 min to 1h.
#Wash beads 5 times with 1 mL Wash Buffer
#Resuspend beads in 50 uL of 5X SDS Cocktail
#For analysis load 10 uL of the resuspended beads and 1% of the lysate. To calculate 0.5% of the lysate, take the volume of lysate added multiply by 0.01, divide it by 5 (because you are only loading 1/5 of the resuspended beads) and multiply by 2 (if using 2X SDS cocktail on the lysate). For example if you loaded the beads with 500 uL lysate, you would run 2uL lysate. It may be necessary to dilute the lysate.

2xHNG Buffer

2012-07-31T17:44:49Z

Sheelak: wrote page

[[ Category:Buffer ]]
[[ Category:Immunoprecipitation]]

{| border="1"
! || Final Concentration || per 100 mL || Stock || Location
|-
| HEPES, pH 7.4 || 100 mM || 10mL || 1M || 4C
|-
| NaCl || 300 mM || 15 mL || 4M || Shelf
|-
| Glycerol || 20% || 20 mL || || Shelf
|}

GST Pulldown Assay

2012-07-31T17:28:46Z

Sheelak: added categories and link to GST-GTPase pull downs

[[ Category: Protein Purification ]]
[[ Category: Protein-Protein Interactions ]]
[[ Category: Protein Biochemistry ]]
[[ Category: Proteins ]]

==Materials==
*2x HNG (100mL). Combine 100mM HEPES (10mL), 300mM NaCl (7.5mL), 20% glycerol (20mL)
*Wash Buffer (50mL). 1x HNG (25mL) and 25 mL water.
*Lysis Buffer (10mL). Combine 5mL 1x HNG, 1 PI tablet, 1mL of 10% NP40 (or 0.5 mL of 20% Triton X-100) and any other cofactors as needed. Bring up to 10 mL in water. This buffer is just a starting point and may need to be modified for particular interactions.
*Glutathione sepharose beads

==Immobilization of Protein onto Glutathione Beads==
#Combine 1 mL PBS (or other buffer if needed) with 200 ug of protein and 40 uL of resuspended gluthione beads.
#Incubate 30min-1h end over end at 4C to bind to beads.
#Centrifuge beads for 30s on high. Aspirate supernatant and add 1 mL PBS (or other buffer)
#Repeat wash step 2x.
#Resuspend beads in ~200 uL final volume of buffer and make 20 uL aliquots (each will contain ~10 ug of protein and will be enough for ~5 pull down assays)
#Freeze these aliquots at -80

==Protocol==
#Prepare lysis buffer with detergent (10 mL) and without detergent (50 mL)
#Prepare and treat cells as needed
#Wash cells 2x with ice cold PBS (10 mL for a 10cm dish, 25 mL for a 15cm dish)
#Add 1 mL lysis buffer and scrape cells with cell scraper. Transfer to 1.5 mL eppendorf tube
#Incubate end over end for 15-30 min at 4C to lyse.
#Centrifuge at 4°C for 10 minutes.
#While waiting for the previous two steps, prepare the affinity beads:
##Thaw one aliquot of immobilized protein and an aliquot of GST.
##Calculate volume of lysate to be used per pulldown. For example if you have >1 mL lysate and you want to do 3 pull downs then you can use up to 333 uL of lysate per pull down. Alternatively you can do a bradford assay and determine the volume of lysate to be used based on the amount of protein. Record how much lysate will be used per pulldown
##Calculate how much buffer to resuspend the beads in. Typically we use one volume of beads per volume of lysate, so if you want to use 300 uL of lysate you would resuspend the beads in 300 uL of buffer. Calculate which combinations of lysate and beads you want to use. Based on the entire tube containing 10 ug, record how much protein is being used for each pulldown. Remember to use the same amount of immobilized protein for each pulldown.
##If necessary aliquot beads into different tubes so that you have one tube for each bead/lysate combination.
#Once lysate is done with centrifugation, transfer the supernatant to a fresh tube.
#Take out a 50 uL aliquot of each lysate sample and add 50 uL of 2x sample buffer as a lysate control.
#Add the appropriate amount of lysate to each set of beads
#Place tubes end over end for 30 min-4h at 4°C. Use 30 min for time sensitive interactions (ie [[GST-GTPase Pull Down Assay]]) and longer time for more stable interactions.
#Add 50µL of glutathione sepharose beads to each tube to increase the bed volume of beads and to limit accidental aspiration of beads
#Wash each tube with 1x HNG five times at 4°C.
#Add 20-40 uL of 2X SDS Loading Dye.
#Boil samples for loading on a gel.
#Calculate what 0.5% Lysate Volume is (remembering it is diluted 2x in sample buffer).
#Load 0.5% Lysate plus the entire volume of the pulldown. If you need to do several blots of the pulldown, decrease both volumes accordingly.