- parts of PAGE apparatus

- two slides are used for gel preparation. put back slide first as shown in figure

- we take 2 spacer in between 2 slides. So gel will be of spacer thickness. to make air tight contact of slides and spacer we use veseline. Apply veseline in both the sides of spacer

- Put the spacers in contact with both end of back slide

- Now put front slide as shown in figure. Make sure both slides and spacer are in air tight contact with each other to avoid leaking of gel

- Put applicator comb in between two slides. Keep slides spacer and comb in such a close approximation that there is no gap in between and gel can not be leaked.

-Now fit the electrode block on both the sides. Fitting screws are behind the apparatus. tighten the screw.

-13 -14

- to avoid leaking of gel from above put a rubber tube coated with veseline , in space in between shown in figure. -17

-put the straight end downward side so gel can not escape from below tube. Alternatively you can use a piece of eraser rubber coated with veseline to fill the space instead of rubber tube -19

Running buffer

Prepare running buffer of pH8.3, 1X as follow 

 tris base= 3.0285gm
 Glycin= 14.4gm
 SDS=1gm
 Make up o 1 liter

1%agarose

prepare 1% agarose in running buffer. 

 Agarose powder= 100mg
 running buffer= 10ml

mix, heat till particles of powder get dissolved and solution become clear. -22 -23

-With the help of pipette pour agarose gel in the front compartment till gel reaches between 2 slides approximately 2mm in lower part.

- pour gel in rubber tube and side of the tube to seal it properly. Allow the gel to cool and solidify.

prepare 10% SDS, 10% APS and TEMED

  1. 10% SDS- dissolve 10gm SDS in 60ml water, heat, mix and dissolve. Make up to 100ml. Make aliquotes of 0.5ml, seal with parafilm and keep frozen.
  2. 10% APS- take 20mg APS in 0.2 ml water. Prepare freshly
  3. TEMED- Take 200ul TEMED in eppendroff cup and keep i sealed to avoid repeated opening of main bottle.

prepare separating gel

  1. Acrylamide-bis- 25%
  2. Target gel- 12.5%
  3. Total Volume- 8ml
  4. Separating buffer, pH 8.8, 4X- 2ml
  5. Acrylamide-bis- 4ml
  6. Water- 2ml
  7. 10% APS- 50ul
  8. TEMED- 10ul
  9. 10% SDS- 80ul

Important note- As TEMED and APS are the main components responsible for polymerization of polyacrylamide gel, add them lastly and only when you are prepared for pouring.(Make sure agarose gel is solidified before pouring polyacrylamide. Preserve small amount(Approx. 1 ml) of separating gel in bottle to check for polymerization.

Remove the applicator comb and pour the separating gel with the help of pipette. -32

After pour of separating gel, add 0.5ml of Water saturated with Butanol over the surface of separating gel With the use of hemilton syringe very slowly(to prevent exposure of separating gel with oxygen). Allow the separation gel to polymerize. -34

stacking gel

After polymerization of separating gel remove butanol with use of hemilton syringe. Prepare stacking gel untill the separating gel get polymerise.

  1. Acrylamide-bis- 25%
  2. Target gel- 4%
  3. Total Volume- 8ml
  4. Stacking buffer, pH 6.8, 4X- 2ml
  5. Acrylamide-bis- 1.28ml
  6. Water- 4.7ml
  7. 10% APS- 50ul
  8. TEMED- 10ul
  9. 10% SDS- 80ul

Important note- As TEMED and APS are the main components responsible for polymerization of polyacrylamide gel, add them lastly and only when you are prepared for pouring.(After butanol have been removed)

Pour stacking gel with pipette up to the upper end of back slide.

Put comb in between 2 slides. some of the stacking gel will overflow on inserting comb. Avoid production of bubble beneath comb.

Allow gel to polymerize

Sample preparation

  1. serum- 1:10 dilution with DI water. Serum and Loading Buffer in ratio of 1:1
  2. CSF- No dilution. Serum and Loading Buffer in ratio of 1:1
  3. E.Coli-Bring E.Coli colonies on agar plate from microbiology department. Take only colony with tip, no agarose by superficial scraping. Keep to tip with E.Coli scratch in testtube. Add 300ul 10% SDS. Heat for 5 min. Add loading buffer in ratio if 1:1.

-42 -43 -44

Now remove the Applicaor comb. Remove bubbles or residual water from well with the help of hemilton syring. -46

Pour running buffer in back compartment till buffer cover the whole gel and wells.

Apply approx.50ul of sample in each well with hemilton syring. -49 -50

Attach with power supply. set the voltage to 90. Run electrophoresis till the -52