Isoelectric focusing (IEF)

IEF modified from Chambers et al., JCB, 2012

for mammalian BiP

Reagents

17.5:1 Acrylamide:Bis working stock (30%)

20 % CHAPS working solution

Urea (highest grade available)

Pharmalytes pH 4.5-5.5 (GE)

APS 10 %

TEMED

1M stock DTT 

200 mM Imidodiphosphate working stock

Sodium pyrophosphate

TBS 1x stock

10 mM Glutamic acid

50 mM Histidine

IllustraÔ AutoSeq G-50 Dye terminator spin columns (GE Healthcare)

Gel Recipe – 2x 0.75 mm Mini gels

-6.34 g Urea

-1.5 ml acrylamide mix

-0.75 ml 20% CHAPS stock

-3.64 ml MilliQ water

        Mix at 30°C until urea dissolved (do not heat higher)

-600 µl Pharmalytes

-30 µl APS

-12 µl TEMED

Pour gels and leave to polymerise for 1 hour (better 3 h). Always use the same day.

Lysis Buffer – 8.8M urea – for 1ml of buffer…

-0.53 g Urea

-4.46mg sodium pyrophosphate

-250 µl 20% CHAPS stock

-10 µl imidodiphosphate 200mM stock

-add MilliQ water to ~900µl

        Mix at 30°C until urea dissolved (do not heat higher)

-50 µl  DTT 1M stock

-20 µl Pharmalytes

        Water to final volume of 1 ml

IEF overlay buffer – aliquot and snap freeze for storage at -80°C

0.75 g urea

50 µl Pharmalytes

MilliQ to 2.5 ml

        Mix at 30°C until urea dissolved

Add small amount of bromophenol blue before freezing

IEF Sample Buffer – for 1ml of buffer…

-0.48 g Urea

-250 µl 20% CHAPS stock

-add MilliQ water to ~900µl

        Mix at 30°C until urea dissolved

-50 µl  DTT 1M stock

-20 µl Pharmalytes

        Water to final volume of 1 ml

IEF Transfer Buffer 

-25 mM Tris, pH 9.2

-190 mM Glycine

-0.01% SDS

-10 % Metanol

Sample Preparation – from 15 cm dish

• Wash cells with 5ml cold TBS on ice.
• Add 1 ml cold TBS to dish and remove cells using cell lifter at acute angle. Transfer to 1.5 ml eppendorf tube.
• Pellet cells at 400g for 5 minutes at 4°C. remove TBS from cell pellet.
• Allow cell pellet to warm to room temperature (perform all subsequent steps at room temperature or at 30°C) and add lysis buffer to 10x pellet volume. Resuspend pellet by flicking and incubate at room temperature for 5 minutes. 
• Spin lysate at full speed in a bench top centrifuge for 10 min to pellet nucleic acid gloop (this causes pipetting problems if not removed). Transfer supernatant to a fresh tube. [Remarks: The lysate can be stored at this step ON @ RT and processed further the next day. We used lysates even after 2-3 days of storage with good results. We usually did ON storage in this way and skipped the following ultracentrifugation step.]
• Spin lysate at 100,000 g for 1 hour (we use 200 µl tubes in Beckman 42.2Ti rotor) @ 30°C to remove nucleic acids. [Remark: This step is not required. Spin for 0.5-1 h at full-speed in microcentrifuge at RT instead.]
• Pre equilibrate AutoSeq spin column with IEF sample buffer
  • Remove cap on bottom of column and unfasten screw cp by 1 turn. Pen mark the barrel of the column and always have this mark facing the centrifuge spindle during spins.
  • Place in collection tube and spin at 1500g for 1 min to remove column storage buffer. Discard flow through.
  • Apply 300µl IEF sample buffer to column and spin 1500g, 1 min.
  • Discard flow through and repeat.
  • Apply a third 300µl of IEF sample buffer to the column and spin 1500g for 2 min. Place column in fresh collection tube.

• Apply 50 µl of 100000g lysate supernatant to centre of column matrix (apply slowly)
• Spin column at 1500g for 2 min. Retain flow through and discard column.
• Clean outer surface of gel plates to remove Urea crystals. Place gel in cassette sealed with vacuum grease.
• Fill tank with 10 mM glutamic acid (anode buffer) and gel cassette with 50 mM histidine (cathode buffer) immediately before use (buffers must not contaminate each other as this will degrade pH gradient).
• Heat IEF sample to 30°C for 3 minutes and centrifuge for 1 min at full speed in bench-top. 
• Wash out empty gel wells with cathode buffer (this is very important and should be done extensively and carefully! E.g. wash extensively with P1000 and press air into wells several times with a syringe until the bottom of the wells stays clearly and sharply visible). Load 15 µl of IEF sample.
• Overlay sample with 5 µl of IEF overlay buffer by pipetting into the middle of the well (adjust pipette to 7 µl and do not press out the rest when loading).
• If running markers, mix 5 µl IEF marker with 10 µl IEF buffer, load and overlay.
• Fill and overlay empty lanes with IEF buffer/overlay.

Run gel

• 100 V, 10 min
• 250 V, 1 hour
• 300 V, 1 hour
• 500 V, 30 min

• Wash gel 3x 10 min in IEF transfer buffer.
• Charge gel by incubation in 1.5M Tris + 0.4% SDS (SDS-PAGE resolving gel stock buffer) for 2 min, rinse with IEF transfer buffer.
• Transfer gel onto nitrocellulose at 300 mA for 3 hours.

Remarks

Comment: The tricky bit is the transfer process as the gels are very fragile and rip or distort if you handle them too much. I found the trick was to separate the gel plates, use your finger to loosen the well lanes at the top of the gel before washing it off the plate into a 20cm tissue culture dish with a slow stream of transfer buffer. If you try to peel it off with your fingers it will rip and also stretches easily, warping your bands. Once it is washed, drain off the vast majority of the buffer and gently shake the dish until the gel lies flat. then place a filter paper on top of the gel (get it right as it won’t come off again), turn the dish upside-down, and peel off the filter paper which brings a nice, flat, undistorted gel with it. Don't use PVDF for the blot, always nitrocellulose.

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