13 mei 2008
Mice synaptic membranes (SM) isolation protocol
(MD-070925; adapted from OK 051214).
I. Chemicals, buffers and equipment to be prepared in advance:
Stock solutions
2M Sucrose (MW 342.3) 0.5l
Dissolve 342.3g of sucrose in 0.5l of MilliQ (MQ) water by stirring;
Be sure that sucrose dissolves completely and there are no crystals in the bottle; after dissolving check the
volume of the solution, if necessary adjust volume with MQ to 0.5l
This solution can be stored at +4°C and used during 4 months at least, but check before every use that there
is no fungi growth. Advice: store 45ml aliquots in 50ml Falcon tubes at -20°C
500 mM Hepes/NaOH pH 7.4 (MW 238.3) 50 ml
Check if the pH-meter has been calibrated properly with 2 standard solutions before any pH adjustments, if
necessary recalibrate it.
Dissolve 5.96 g of Hepes in 40ml of MQ water, adjust pH to 7.4 with freshly made 3M NaOH (1.2g NaOH in
10ml of MQ water), adjust volume to 50 ml
Working buffers (make one day prior to use)
A. Homogenisation Buffer:
5 mM HEPES/NaOH pH 7.4
0.32M Sucrose
B. 0.85 M Sucrose:
5 mM HEPES/NaOH pH 7.4
0.85M Sucrose
C. 1.2 M Sucrose:
5 mM HEPES/NaOH pH 7.4
1.2M Sucrose
D. The 5 mM HEPES:
5 mM HEPES/NaOH pH 7.4
Buffer
50 ml
300 ml
500 mM Hepes
2 M Sucrose
500 mM Hepes
2 M Sucrose
Homogenisation
0.5 ml
8 ml
3 ml
48 ml
0.85 M Sucrose
0.5 ml
21.25 ml
3 ml
127.5 ml
1.2 M Sucrose
0.5 ml
30 ml
3 ml
180 ml
5 mM Hepes
0.5 ml
-
3 ml
-
6 Samples: 300 ml homogenisation, 100 ml 5mM hepes, 100 ml 0,85 and 1,2M Sucrose.
50 ml buffers can be made in Blue capped Falcon tubes. After preparation keep them at +4°C.
NOTE: check ultracentrifuge availability way in advance and make a reservation (list can be found on top
of the centrifuge). Also put the black rotor buckets at +4°C to save cooling time.
Check clean glassware availability
The isolation day
First things to do:
1. Switch on the low speed Eppendorf centrifuge 5810R (with rotor for 50ml Falcon tubes) and run the
“Fast cooling” program
2. Take ice and put Potter homogeniser (5ml) on it with the Teflon pestle inside
3. Put labelled 50ml/15ml/5 ml Falcon tubes on ice, one for every sample (use 5 ml tubes if you do RNA
isolation
4. Prepare the Homogenisation buffer with inhibitors proteinase en RNAse inhibitor:
Fill a round dish with ice, put a beker in it with (for 6 samples) 50 ml of the Homogenisation buffer, a
stirring bead and add 1 tablet of the Complete EDTA-free Roche inhibitory cocktail (fridge in C321).
NOTE: there are two kinds of Roche tablets the Big one (no sign on the bottle; 11 873 580 001) and
the Mini (called Roche mini tablets; indicate nr). The mini tablets are for 10 ml, for 50 ml 1 big
tablet is enough. Be sure to take the right one! And ad 0.2 µl/ml RNAse inhibitor
5. Take a pair of tweezers (pincers; sharp ends): not all of them can reach the bottom of a 1.5 ml
Eppendorf tube.
When ready and the homogenization buffer is cold, start:
I. Tissue Homogenization (in cold room)
1. Attach the cold Teflon pestle to the Dounce Homogenizer (B. Brown Biotech. International) and set
the homogenizer to 900rpm (do not switch on yet);
2. Pour 5 ml (or 2.5 if you do RNA isolation too) of the homogenization buffer into the Potter (leave the
Potter on ice);
3. Take out a tube with brain tissue from -80°C or from box of dry ice, and drop the piece of tissue
directly into the homogeniser. If necessary, flick the tube or use the forceps to take out the tissue.
Work fast, if the tissue warms up it becomes messy and very difficult to handle;
4. Place the Teflon pestle into the Potter with your sample, and only after that!!! switch on the
homogenizer – at 900 rpm;
5. Homogenize the sample with 12 slow strokes at 900 rpm (moving all the way up and down
without taking the pestle out of the buffer);
6. Carefully transfer the homogenate to the labelled tube;
7. Wash the Potter and its pestle well with MQ and put on ice (with the pestle) for 2-3 min.
8. When cooled down start with the next sample.
IIa. Low speed centrifugation: purification from cell debris
Centrifugation 1000g x 10 min x 4°C
Eppendorf centrifuge 5810R (for Falcon tubes) with swing-out rotor.
Be sure to select rfc (rotation forces), NOT rpm
IIb. RNA isolation
1. Take supernatant S1 (~2 ml in 2 ml tubes) (Fig 1.) and transfer to labelled tubes.
2. Resuspend palet P1 in 2 ml homogenization buffer and again centrifuge 1000g x 10 min x 4°C
3. Take supernatant (S2). Let pellet P2 leak (tube upside down) and store P2 in -20 °C for RNA
isolation.
4. centrifuge S1 and S2 20000g x 10 min x 4°C (in 4 * 2ml tubes on table top),
5. Store Supernatant S3.1 in -20 °C for RNA isolation and resuspend palet (P3) in 5 ml homogenization
buffer (by pipetting up and down (1 ml pipet) and load on gradient (S3.2 can be discarded, hardly
RNA anyway)
6. The next day or during one of ultra centrifugation steps, split S3.1 over six 1,5 ml epjes (NOT
2ml, because you don’t get a palet in these tubes!!!!) and ad 600 ul Tri-reagent to S3.1
fractions and add 600 ul Tri-reagent to P2 and grind and pipet to resuspend palet. Add 120 ul
chloroform to each sample.
•spin 10 min at 14,000 g (4° C), stop centrifuge in ‘slow mode’, so you don’t diturb the
interphase. The upper phase contains the RNA, the interphase proteins and genomic
DNA.
•Transfer the upper phase to a new 1.5 ml tube, be careful to not to take the
interphase.
• Perform a Chloroform extraction at this stage (see chloroform extraction at
DNAsetreatment), and to continue with the steps below.
• Add 2 ul glycogen and add 0.9 volumes of isopropanol, shake to mix and leave on ice
for 20-30 min (do not put in the freezer!)> With low amount of RNA ad 2 ul of glycogen
(2 ug/ul) as a co-precipitant.
•Spin 15 min at 14,000 g (4° C).
•Take off supernatant (trash), don’t disturb the pellet. And Wash once with 96%
ethanol to remove isopropanol and spin shortly (2 min) and remove sup
•Wash with ~100 µl 70% EtOH, spin shortly to get the pellet at the bottom.
•Remove all supernatant and dry the pellet at RT (~10 min).
•Resuspend the palets of S3.1 fractions in ~100 ul total (pool them in one fraction).
And add -50 ul to P2 RNA fraction. Store both RNA fractions in -80 C.
Figure 1.
III. UltracentrifugationI: 0.85-1.2M sucrose step gradient:
1. While the samples are being centrifuged, establish a step gradient in tubes for ultracentrifugation:
Sample volume
Centrifuge buckets
Buffers
Sample volume
≤ 5ml
‘small’ (16ml total)
5ml 1.2M
5-5.5ml
5ml 0.85M
> 5 – 17ml
‘big’ (37ml total)
12ml 1.2M
15-17ml
12ml 0.85M
Take ~17 ml ultracentrifugation tubes from the cabinet next to the door to lab C321a. The sample
volume is ~5 ml so:
Pipette (volume pipette) 5 ml 1.2M Sucrose into tube
Pipette (carefully and slowly, while keeping tube under an angle!) 5 ml 0.85M Sucrose on top
to get two clear phases
2. After making the gradient, put tube on ice. Take tubes with sample from low speed centrifuge and
put them on ice.
3. When transferring the supernatant try not to touch the bottom of the tube with your pipette tip. Very
carefully put the supernatant on top of the sucrose step gradient (~5.5 ml).
4. Be sure that between the surface level of the solution and the edges of the tube a maximum of 5-7
mm space is left (if not - adjust the level with the Homogenisation buffer), otherwise edges of the
tube may collapse in the ultracentrifuge
5. Balance buckets with tubes on a balance (with MQ). The difference between two opposite tubes (1-4;
2-5; 3-6) should not exceed 0.02g!! When using 4 tubes, leave the two remaining (coupled =
opposite!) buckets empty, but do put them in the centrifuge
6. Centrifuge at 30.000rpm x 2h x 4°C
7. Collect the white disk of the 1.2-0.85M interphase (=synaptosomes), using a 1 ml pipet with a yellow
tip on a white tip.
Advice: First use a plastic Pasteur pipette to take off from the top the stuff you don’t want (leave a bit).
Then take a fresh pipette to carefully collect the white disk at the interphase and transfer to a fresh
(17ml) ultracentrifuge tube (alternatively it can be transferred to a Falcon tube and frozen to continue
later).
IV. UltracentrifugationII: SMP pelletting
1. To the collected sample (~ 2 ml), add an equal volume (~2 ml) 5 mM HEPES and fill up to ~15ml
with homogenisation buffer, carefully mix it by pipetting (10 times);
2. Balance (red) buckets with tubes on a balance. The difference between two opposite tubes (1-4; 2-5;
3-6) should not exceed 0.02g!!
3. Centrifuge the samples at 25000rpm x 30 min x 4°C
4. Meanwhile:
Dissolve Mini Roche in 5 mM HEPES (1 tablet/10 ml) for 4 samples 2 tablets in 25 ml; for 6 samples 3
tablets in 35 ml.
Take Boehringer box or the lid of it, fill with ice and put it on the VarioMac Multipoint multiple stirrer.
Position small glass vials + magnetic stirring bar onto the ice, on stirring spots
5. Take off the supernatant until the belt of the centrifuge tube (just pour off and leave a bit) (~0.5 ml
solution on the bottom left);
6. Resuspend the pellet by pipetting (1 ml “blue tip”) at least 20 times
NOTE: During all these manipulations leave the tubes on ice
V. Osmotic shock of SMPs
1. Transfer the resuspended SMPs to respective precooled glass vials
2. Wash the tube with 5ml 5mM HEPES (with Roche inhibitor) and add this 5ml to the respective glass
vial on ice, to yield a total of ~5.5ml. Make sure no pellet it left
3. Stir at 250rpm x 15min on ice
VI. UltracentrifugationIII: 0.85-1.2M sucrose step gradient:
1. While the SMPs are shocking, prepare a 0.85-1.2M Sucrose step gradient (use 5ml 1.2M; then
carefully put 5ml 0.85M on top)
2. Carefully put the shocked SMPs (~5.5ml) on top of the sucrose step gradient.
3. Balance buckets with tubes on a balance. The difference between two opposite tubes (1-4; 2-5; 3-6)
should not exceed 0.02g!!
4. Centrifuge at 30.000rpm x 2h x 4°C
5. Collect the white disk of the 1.2-0.85M interphase in a ultracentrifuge tube.
VII. UltracentrifugationIV: SM peletting
1. To the collected sample, add 5 mM HEPES up to ~15ml, carefully mix it by pipetting (10 times);
2. Balance buckets with tubes on a balance. The difference between two opposite tubes (1-4; 2-5; 3-6)
should not exceed 0.02g!!
3. Centrifuge the samples at 25000rpm x 30 min x 4°C
4. Take off the supernatant until the belt of the centrifuge tube (leave ~0.5 ml solution);
5. - For westernblotting: Resuspend the pellet by pipetting (1 ml “blue tip”) at least 20 times. During
all manipulations leave tubes on ice; Be sure to wash the pellet completely.
- For SM: Take off all supernatant with 1 ml tip and resuspend in ~70 ul Hepes using a 1 ml tip
(because of low amount of protein).
6. Transfer the sample to a 1.5 ml Eppendorf tube;
7. Store at -20°C or continue with the protein determination.
VIII. Protein determination
1. BSA will be used as a reference protein to make a ‘ladder’ (concentration series).
Take one tube with 50l BSA (1 or 2mg/ml) stock from the freezer and defrost
2. Take the bottle of Bradford from the fridge (BioRad Protein Assay; dye reagent concentrate; cat#
500-0006; München DE; 4°C)
Per sample 1ml of a 1:4 Bradford:H2O mixture is required (I used 16 samples, so 16ml), plus 7ml for
the 7 resp. references. In this case I made 30ml (24ml H2O + 6ml Bradford);
3. Make the 7 reference concentrations, according to this schematic:
Bradford-H2O mixture
BSA-HEPES mixture
end concentration (g/ml)
1000 l
0 l
0
1000 l
1 l
1
1000 l
3 l
3
1000 l
5 l
5
1000 l
10 l
10
1000 l
15 l
15
1000 l
20 l
20
4. Ad 1 or 2 ul of each sample to the Bradford (or more if you resuspended the palet in more than 70
ul) (BSA standard is 1 or 2 ug/ul  concentration is the same as BSA)
5. Ad all samples to plastic cuvette and incubate for 10 min (1h max.)
6. On the Eppendorf BioPhotometer take biaorad assay, measure standard without BSA as blank and
then first establish the control concentration ladder, then measure the protein samples
7. Determine concentration by the trend line of your standards
8. Load ~ 2 ug on SDS gel and tain with colloidal coomassie. Correct the protein concentrations based
on coomassie intensity for each sample and the average coomassie staining (see excel sheet)
IX. Final SM preparation
1. Using the determined protein concentrations, transfer 75 g to a fresh eppendorf tube and label it;
2. Speedvac (~3h.) to dry and store at -80°C