Esophageal Adenocarcinoma Methods and Protocols

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This technique is as an alternative to anion exchange chromatogra-

phy. Isoelectric focusing separates proteins according to their pI

(isoelectric point) on immobilized pH gradient (IPG) strips. The

off-gel fractionator is designed to allow the proteins to be collected

in the liquid phase in multi-well compartments mounted over the

IPG strips allowing ease of collection of fractionated proteins for

further analysis.

1. Place a 24 cm IPG strip in a tray and a 24-well frame on top.
   Rehydrate the IPG strip using the off-gel separation buffer and
   pads placed at each end of the strip.


2. Add sample diluted in off-gel separation buffer to each well,
   add a cover seal, and place mineral oil at each end of the strip.


3. Apply a current for 36–48 h (typical initial voltage
   200–1500 V).


4. Remove sample fractions from each well and reconcentrate
   into desalt buffer using spin column filters.

Further fractionation by size is performed using SDS-polyacrylamide

gel electrophoresis (SDS-PAGE) using conditions selected to

achieve optimal separation of lower molecular weight bands.

1. Add the desalted fractions to sample loading buffer, reduce in
   10 mM dithiothreitol (10 min incubation at 70 °C), and alkyl-
   ate in 50 mM iodoacetamide (30 min incubation at room
   temperature).


2. Load the samples onto the gel with MES running buffer. Run
   pre-stained standards in the marker lanes. Perform electropho-
   resis at 200 V for 35–40 min. Fix the gels with 50% metha-
   nol/10% acetic acid and stain with Coomassie G-250 colloidal
   stain (Invitrogen) for 3–12 h. The colloidal blue stain utilizes
   colloidal chemistry that reduces free dye in solution and
   improves protein-to-dye binding ratios to improve sensitivity.


3. Destain with deionized water overnight.


4. Excise selected protein bands, according to molecular weight
   from gels using a Harris Unicore 1 mm cutter and store in
   Eppendorf tubes at − 20 °C prior to identification (see
   Note 10 ).


5. To confirm the presence of the peak of interest in the excised
   band by SELDI-TOF MS, passively elute proteins from the
   excised bands as follows:
   (a) Wash with stain remover.
   (b) Dehydrated using acetonitrile.
   (c) Elute proteins with elution buffer by overnight incubation
   and sonication.

3.3.2 Fractionate
by Charge

3.3.3 Further Fractionate
by Size

3.3.4 Collect Fractions
for Identification

Proteomic Protocol