41


Table 2. Rehydration of IPG Strips CONCLUSION


The Hoefer IEF100 offers a simple and fast means of separating proteins. No prior experience running 2D equipment is needed to operate the IEF100.


The preprogrammed protocols make running the unit easy, furthermore, the programs are easily edited to support flexibility as needs evolve.


An impressive feature of the IEF100 unit is its unique ability to monitor each IPG strip individually in real time and maintain a record of each strip’s focusing data for later review and publishing. This avoids wasting money and time on the second dimension separation of IPG strips that did not focus correctly in the first dimension.


The IEF100’s ability to run at 12,000V, more than three times other competitive units, enables 2D separation in a single day.


Our assessment of the IEF100 is that it is extremely user friendly and supports easy and accurate separation of proteins of similar molecular weights.


IPG Strip Length 7cm 18cm 24cm Volume per Strip (µl) Reagent 130 340 450 6M Urea 2% SDS Prepare immediately prior to use


Concentration Range


3.6g/10ml 0.2g/10ml 0.375M Tris-HCl 2.5ml 1.5M


20% Glycerol 135mM


Iodoacetamide


Table 3. Preset Program parameters in the Hoefer IEF100


Program 2


Name: 7cm Constant watt Delay 0:00, Delay temp 20°C, Run temp 20°C, 500 µA, 6000 V, 0.5 W


Table 1. Rehydration Buffer Reagent Urea* CHAPS DTT Carrier


Concentration Range


8M (8-9M) 1% (1-4%) Amount 4.8g Table 4. Equilibration Buffer I (EBI) TEMED 10mg Prepare immediately prior to use 13mM (13-100mM) 20mg Ampholytes, 40% 0.5% (0.25-2%) Distilled De-Ionized Water *Urea can be replaced with up to 25% Thiourea


When DTT is used, a second equilibration step is required. The Iodoacetamide prevents protein re-oxidation during electrophoresis and alkylates residual DTT to minimise vertical streaking.


125µl 10ml 0.375M Tris-HCl 2.5ml 1.5M 20% Glycerol


Iodoacetamide 200mg/10ml pH 8.8 for the 10ml mixture


130mM 2ml/10ml 20mg 125µl 10ml Reagent 6M Urea 2% SDS


Concentration Range


3.6g/10ml 0.2g/10ml Amount 4.8g 10mg Distilled Water


30% Stock Acrylamide Solution


4 X Tris-Glycine Solution pH 8.8


10% SDS


10% Ammonium Persulphate


TEMED Table 7. 4% Acrylamide solution stacking gel 4% Acrylamide gel 6.1ml 1.3ml 2.5ml 100µl 50µl 10µl 15µl


Step 1, Constant watt, 0.1W, 1:00 Hrs Step 2, Constant watt, 0.5W, 8000 Vhrs Step 3, Constant volt, 1000 V, 1:00 Hrs


Distilled Water


30% Stock Acrylamide Solution


4 X Tris-Glycine Solution pH 8.8/ 0.4% SDS


10% Ammonium Persulphate


2ml/10ml 250mg/10ml Amount 4.8g 10mg 20mg 125µl 10ml


pH 8.8 for the 10ml mixture Table 6. 10% Acrylamide solution resolving gel


10% Acrylamide gel 6.3ml 5ml 3.75ml 150µl


Table 5. Equilibration Buffer II (EBII)


New Validated Cell Colony Formation Assay TTP LabTech has validated a new cell colony formation assay on the Acumen®


this latest protocol uses fixed as opposed to live cell colonies for analysis.


The Acumen eX3 laser scanning cytometer is an ideal platform on which to analyse cell colony formation assays. It enables rapid and accurate enumeration of colony number and is more suitable for higher throughput compound assessment than current microscope based methods. This non-confocal system enables scanning of whole wells for fluorescent objects without the need for the nuclear stains as required by other platforms. This approach determines colony number through the application of a volume algorithm and permits the differentiation of cytostatic effects, where the number of colonies and size remains constant, from cytotoxic effects where the size and number may be reduced. Application of Acumen’s microplate cytometric technology offers significant benefits over alternative methods in the search for novel chemotherapeutic agents and provides a simple high content and high throughput assay.


Circle no. 150


eX3 fluorescence microplate cytometer in collaboration with ChemPartner. As an alternative to the traditional method


Spotlight


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