PUT UP THE SYSTEM
- Glasses: clean thoroughly (distilled water, alcohol) + dry well;
- Place glass plates in holder (check if bottom glasses are perfectly parallel), fix & place in rack.
- Fill system with water to check for leaks. If fine pour off water and then pour gel
MAKE RUNNING GEL (45 min to 1hr)
- For two mini gel- 10ml resolving, 5ml stacking.
- Find out optimal gel concentration for your protein of interest.
- 150-250 kDa 7%
- 75-150 kDa 8%
- 35-75 kDa 10%
- <35 kDa 12%
- Mix well but carefully to avoid bubbles after adding every ingredient.
- Add APS and TEMED in the end; TEMED is always last as it initiates the polymerization!
- Pipette gel between glass plates and the optimal leave after it solidified is ± 1 cm space below bottom comb.
- Cover carefully with water (to ensure a smooth gel surface). Gel will smooth out with time.
- Let it stand undisturbed for 30-40min. Take an idea whether gel has solidified or not by checking the leftover solution in the tube.
- When running gel is made O/N: pour off water next morning and acclimatize the gel for 10 min at RT.
- If you want to preserve the gel longer later use (only possible for running gel), then cover with wet tissues and saran wrap and leave in the 4oC.
- Use the time in b/w to prepare your lysates
DENATURE SAMPLES
- For lysate preparation- use 5X sample buffer so: e.g. 80 µl sample + 20µl buffer.
- Mix well. Spin down samples.
- Place sample tubes in 100oC heating block (something heavy can be put on top to prevent the tubes from popping out).
- Time for 5 minutes.
- Put samples back on ice and spin shortly to get condensate down.
- Marker- If using NEB marker heat for 1-2min (kept in -20 oC).
MAKE STACKING GEL (APPROX. 15-30 MIN)
- Pour off water.
- Make stacking gel and pour until space between the glasses is totally filled, put the comb at an angle, this prevents air bubbles.
- Let the gel solidify for 15-20min.
- Remove green comb straight upwards carefully.
- Remove the glass plate assembly from the clamps.
- Wash the assembly gel (the gel with glass plates) under running distilled water with the wells down. Wash the wells if you see any gel pieces inside.
LOADING SAMPLES AND RUNNING THE GEL
- Create the inner chamber of gel tank by putting two gel plates together with the electrode assembly. Make sure the wells are facing IN.
- Make 1x running buffer and fill the assembly. Inner chamber completely to fill the wells. Ensure there isn’t any leaking.
- Outer chamber till the mark
- Use 6 µl of Ladders and carefully load samples.
- Place the lid and start
TRANSFER
- Make transfer buffer. Better to use chilled buffer.
- Cut PVDF membrane in size of 8.5 by 5.7 cm per gel (with gloves).
- Write date, gel name, Ab name, your initial with a gel pen.
- Put in methanol for 5-10 min (to activate) then move to transfer buffer (~2 min). Save methanol.
- Soak sponge (2 per gel), filter pads (2 per gel) in transfer buffer. Remove the bubbles from the sponge using roller gently.
- Use gel releaser/spacer to open plates carefully. Scrape off the stacking gel.
- To remove the gel from glass plate, put the plate in the transfer buffer on top of the membrane (gel side down) and move up and down gently for the gel to detach.
- Preparation of transfer sandwich in gel holder cassette- order MUST be followed!!
- Close the cassette carefully.
- Put the cassettes in the tank (black to black).
- Put a stirrer and cooling block (kept at-30 oC).
- Add transfer buffer till the mark, put it on stirrer.
- Run at 60V, 340mAmp for 2-3hr (constant vol) (mAmp shld be <150).
- If O/N run at 30V, 80mAmp (constant vol).
STAINING AND IMAGING
- Make PBS with Tween= PBST
- Remove blot and place it in a right sized box.
- Add Ponceau S dye (to check transfer) enough to cover the blot.
BLOCK
- Incubate blot 1 hour at RT to block. Blocking mix= for mini gel 10ml PBST and 0.5gm milk (5% non-fat-dry milk powder/BSA).
- Close the milk bottle tight. Do not use if it has caught moisture.
- Note: some 1st Abs does not bind well after blocking with 5% milk. Alternative is 5% BSA.
PRIMARY ANTIBODY
- Discard blocking and add primary antibody..
- Incubate O/N as required. O/N is at 4 oC always.
- Note: some antibodies are preferred to be diluted in BSA, check the datasheet of the antibody! Then also block in BSA!
- Save primary antibody in a falcon tube and store at -20 oC. Note down the number of times it has been used on the tube. Some antibodies can be reused 4-5 times. Check for your antibody.
Secondary ANTIBODY
- Wash 3 x 5 minutes with PBST.
- Depending on the species in which primary antibody has been synthesized use secondary antibody. 1:5000 (mouse); 1:2500 (rabbit) in 10 ml milk /PBST for 1 hour at RT.
- Always throw away 2ndAb (HRP not stable).
- Wash 3 x 5 minutes with PBST.
- Now blot ready to be imaged (Do not stop at this step, finish imaging).
IMAGING
- Put ON the Imaging system. It takes 5-10 mins for the camera to cool down.
- Open Imaging software.
- You will need 0.6ml ECL per blot.
- To make ECL= mix component A and B in 1:1 ratio (so for one mini blot 300ul A + 300ul B). Make sure the fluids stay pure (so use clean pipette tips for every bottle).
- Drain the excess buffer from the blot by touching the edges on a tissue wipe, and then place the blot on a plastic sheet.
- Put ECL on the blot sufficient to cover it completely.
- Leave OPEN and Let incubate for 2 minutes.
- Drain the excess buffer from the blot.
- Put the blot in-between a plastic sheet and remove the bubbles.
- Put the blot on black tray in the centre. Put it inside the system (the side of the tray with holes facing out). Make sure you push the tray to touch the sensor.
- Adjust Focus, START.
- Once done, save the images.
- Capture a white light image for aligning with the marker (do not move the blot position).
