2D-IEF protocol

4/9/01

Before starting:

PREPARE SAMPLES

We are doing 1 10cm 293T cells in 1.2mL sample buffer, 1 10cm (or equivalent = 6 6 wells, 12 12wells) in 300 or 600uL of sample buffer

Test your samples for protein expression (and for P-CREB to test induction) by western

Samples should not be warmed or boiled

Thaw at RT, cannot keep on ice or urea will come out

Sample Buffer:

9.5M urea (14.25g)

2% Tx-100 (5.0mL 10%)

2% Pharmalyte 3-10 (0.5mL)

500mg DTT

35mg PMSF

make to 25mL

plus few grains bromphenol blue to color

Day 1:

SWELL STRIPS, AND SWELL PROTEIN INTO STRIPS

You need the reswelling tray, the mineral oil, the strips (in CWC –20) and the samples, spun down.

Choosing a strip: choices are big (18cm) or small (11cm), 3-10L or NL(lg only), 4-7, 6-11

Balance the swelling tray with the bubble. Make sure lanes are clean

Tape tray in place

Place clean paper on bench, remove strips with clean forceps, gloves and place on paper

Use your finger to determine which side at the arrow end has flimsy plastic

Label the other side with pen.

Distribute 200uL sample along the well

Remove plastic and place strip face down in sample no bubbles, hold arrow end

Cover right away with 3mL mineral oil, then do next sample

Screw cover on strip tray and leave O/N to swell. Time about 24 hours best.

Day 2:

FOCUS STRIPS

Strip focussing time for planning is 20.5 hrs for big strips, 16.5-17.5 hours for small. Start near end of day to have done at a convenient time next day. We start 6pm, running by 6:20, will finish 11:20am next day.

Turn on water circulator below bench. Set mode to “S” for set, make sure set to 20. Hit enter, then set mode to “ “ will read current temp.

Turn on flow valve at gel appartus on bench so water flows through. Arrow should point across the joint to the apparatus.

Level gel apparatus with turnable feet. Use a bubble to make sure its level.

Clean off strip plate “immobiline strip tray” and plastic insert tray (from “stuff” box) with soapy water, rinse, and 70% EtOH every time you use. Wait for them to air dry completely. Get out the two clamp-on electrodes from the box, red and black.

Clean off ceramic plate on gel apparatus with paper towel.

Put a clean piece of 3MM paper on the bench.

Get the pack of long thin 3MM strips from the stuff box.

Remove 1 onto the paper on bench and cut with a clean razor blade to 11cm.

Get a tube of fresh milliQ water.

Use 500uL ddH2O to wet each 3MM strip, by dripping on the strip. Let absorb for several minutes. Note it is key to have as little excess water as possible through the remaining steps.

Add 5mL mineral oil (“Dry strip cover fluid”) to ceramic plate in zig-zag over center portion.

Place the “immobiline strip tray” down over the oil, squishing the oil as you go.

Put 10mL oil over the top of the glass on the “immobiline strip tray”.

Put the plastic strip holder over the tray with the concave face up (strips will fit into the concave spaces) Bubbles under the plastic don’t matter as the concave parts will have good contact. However, don’t squish so hard you get oil over onto the top of the plastic sheet.

Fill a bucket with 1L milliQ water.

Open the tray with the swelled strips from yesterday.

Grasp the strips at the arrow end with forceps, and rinse by dunking in the bucket of water.

Dry the plastic back on the 3MM paper, then rest the strip on its side edge to let water drain (do carefully so strip doesn’t fall over with the gel side down on the paper). Let sit on side for 2’.

Place the strip gel side up, and arrow end pointing toward you in a groove of the plastic surface of the “immobiline strip tray”. Strips should be in the middle of the plate, and line up the ends of all strips.

Use a kimwipe to dab excess water from the 3MM strips, then lay these strips parallel to one another, perpendicular to the gel strips across the top and bottom ends of the gel strips. The 3MM strips should directly contact the gel on each end, make sure they aren’t just across the plastic on the arrow end.

The electrodes are clamped on over the 3MM strips with the red on the bottom, hooked on the L side, the black on the top, hooked on the R side. Make sure the wire of the electrode is down tightly on the 3MM strip, and make sure the electrodes overlap the metal strip on the side of the gel apparatus.

Now pour oil all over the strips and the plastic plate to completely cover it.

Put the cover on, and plug into the power supply (EVS 3500XL “Kunkel”)

Turn power supply on in back on R, scroll arrows to preset “3” (pg. 23 in book), exit, then run. Using 1 extra hour on last step for extra amphilytes (pharmalytes) in sample buffer, therefore program runs for 17.5 hours. Expect current of 0.5-several milliAmps. If you see 0mA, then you have poor connection between the electrodes and the 3MM strips.

Day3:

RUN SECOND DIMENSION IN GEL

You should be able to see the BPB near the arrow end of the strip now a yellow color and focussed in a largish spot.

Reset the cooler to 16 degrees (hit enter…)

Prepare the gel equilibration buffer for the strips.

need 40mL for two strips

Pour 12mL glycerol in a tube

Add 14.4g urea

Add 2mL 1M Tris pH6.8

Add water to 36 mL

Shake to get in solution

Then add 4 mL 10% SDS (1% final)

In two other 50mL tubes measure out 200mg DTT in one (-20 dessicated), and 0.9 g iodoacetimide (CWC 4 degree). Add 20mL equil buffer to each and get in solution. To iodo add BPB to color.

Get the gel and buffer strips. 4-18% kept at RT in the stuff box. 1 gel for 2 11cm strips. Some other gels kept on CWC 4 degree shelf in cold room. Buffer strips made of gel need one + (yellow) and one – (clear) are kept in cold room CWC shelf.

Get a 1L bucket filled with ddH2O

Petri dish for each strip, put 10mL DTT solution in each.

Turn off power supply, take apparatus apart, remove wicks, dunk gel strips to get excess oil off. Then place strip in petri dish with plastic against the side, curved toward liquid. Watch to make sure the strip doesn’t pop out as you are bending it. Slow shake for 10’.

Pipette off the DTT solution and replace with 10mL IA solution. Slow shake 10’.

Prepare apparatus for gel: take strip holder off and wash. Wipe off ceramic plate. Rebalance gel setup. Put new 2.5mL oil in center of ceramic plate.

Open gel cutting edges of package. The gel is fully protected on both sides at this point. Note that arrows on the gel point to + side which is L. There is a notch in the upper L corner as you hold the gel with arrows L.

Lay gel on oil and squish out. Then peel off plastic cover.

Lay buffer strips over the edges of the gel with smaller side of the wedge facing down. Use forceps to push out air bubbles. Yellow is + and goes on the L, clear is – and goes on the R.

Take out 5 small whatman sample app. pieces. 2 are per strip, 1 for the + control and St.

Pull the strips out from the IA solution dish. Put on its back, then on its side on 3MM paper to dry.

Place strips gel side down onto gel with arrow pointed toward the bottom. Push strips to the top and bottom edges of the gel. Tap down gently to remove bubbles. The strips should be about 1mm below the buffer strip on the R, but not directly touching. Place sample application pieces long side L-R under each end of the strip.

Between the strips place the standard app. piece. For standards/+control, mix 10uL rainbow standards with 2uL + control (TNT for me) and spot on sample app. piece. Cut about 1/3 off piece in long dimension, then align between the strips, not touching.

Place thinner gel ap. cover over the gel and adjust electrodes so that they are directly over the buffer strips. Plug in the electrodes to the little holes.

Add the gel ap. lid.

Run program 1. When it starts, amps should be about 20mA for 30’. At this point, pause the cycle, open the app. and remove the strips and the sample app. pieces (dye should have migrated well out of the strips). Scrunch the R buffer strip down to the L a bit, adjust electrode position. Close up gel box again and complete run (1h 45m).

When program stops, get gel off: Get out the white gel cutting box that looks like a tennis bubble. Take the gel off the box, and wash the oil off the back of the gel carefully under running water in the sink.

Put the gel on a hard surface, gel side up, and use a razor to cut top and bottom of gel (stack, and below dye front) and split in half across standards. Notch lower R edge of each gel.

Put gel on slider box with the gel side up. Hook up the wire at one end using the metal clamps on each side. Slide the wire under the gel from one end to the other to release. Take off the top and bottom of the gel.

Use wet 3MM (in transfer buffer) cut to gel size. Place over gel, then flip gel over and pull plastic away from gel to drop onto 3MM. Place gel side up in transfer box, and cover with wetted PVDF. Very gently push out bubbles with your gloved hands or if needed, really carefully roll over a 5mL pipette. More 3mM paper on top.

Xfer 12V for 90’ (for 96kD CaRF protein from 8-18% gel).

Notes:

All the stuff is kept over AB’s bench in the box marked “isoelectric focussing stuff”

Clean things with soap and water, rinse well to rid soap, rinse ddH2O, milliQ, 70% EtOH