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Gel Electrophoresis & Western Blot Protocol

By Michelle Graham,2014-06-06 10:26
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Gel Electrophoresis & Western Blot Protocol

    SKG 8/4/10

    Gel Electrophoresis & Western Blot Protocol

    Materials:

Invitrogen SureLock Mini Cell (chamber, lid, lock, dummy gel, electrode gel-

    running cassette, Western Blot Casette)Invitrogen Power Supply

    “BSA-TBS Tween” beaker Running buffer (4C)

    “TBS Tween only” graduated Large tray

    cylinder Ice & ice bucket

    stir bars Scissors

    strip box diHO 2

    ruler or straight edge Novex Molecular Weight Marker (-

    clean razor blade 20C)

    clean surface to cut membrane LDS Sample Buffer

    (plastic lid) Gel loading tips (at least 15 per gel)

    1 Ab solutions (4C) Pipettes

    rocker 4 sponge pads (per gel)

    2 Ab (4C) (2.5 ;L/strip) Transfer buffer (4C)

    angled tweezers “Transfer buffer only” dish

    15mL conicals gel knife

    1.5 mL eppendorfs paper towels

    ECL Developing kit (and ECL+ if “methanol only” dish

    necessary) methanol (in hood)

    Autoradiography Cassette filter paper

    Autoradiography Film PVDF membrane (or nitrocellulose)

    Plastic transparent sheet protectors NuPAGE reducing agent (4C)

    Timers Colored tapes

    Film developer Apex nonfat dry milk

    Stripping solution TBS-Tween 20 (lots)

    Transfer pipettes BSA (if making antibody solutions) “western blotting milk only” beaker

Procedure

    *note lot # for all reagents*

Sample Prep for Running Gels

    1. calculating required amount of each reagent using excel sheet

    a. get protein assay data and excel sheet

    b. Use data from “DATA WITH BLANK SUBTRACTED”

    c. enter BSA concentrations from least to greatest (.156, .3125, etc.) stndrdd. enter in each triplicate value under (1, 2, 3 measure)

    e. enter title of samples under “NRVM samples” – (10 kPa A, etc)

    i. make sure the “average” column is taking values from all 3

    triplicates

    f. set amount of sample you want to load per well (each well holds 37

    ;L, so do 30;L max)

    SKG 8/4/10

    Gel Electrophoresis & Western Blot Protocol

    g. if you made dilutions for your assay, multiply “calculated values”

    column by the reciprocal of your dilution. For ex, if you diluted your

    samples 1:1, you reduced the concentration by ?. So, multiply

    “calculated values” by 2.

    h. if the calculation is telling you to add a large amount of sample per

    well, that means your lysate is too dilute

    2. label Eppendorf tubes

    3. turn on hot plate to 70C (should be marked with tape)

    4. get proteins out on ice

    5. add indicated amount of LDS (invert solution to mix before using) 6. add NuPAGE reducing agent (use new tip each time)

    7. add in sample (vortex first!) & flush-mix

    8. close tubes and lock top with eppendorf cappers

    9. place samples in heat block for 10 min

    10. remove samples and keep at room temp until loading (store in -20C if not

    running gel immediately after)

    Day 1

    Gel Electrophoresis

    1. put running buffer in freezer (~30 min) to get extra cold while you set up 2. Assemble chamber

    a. Make sure rubber gaskets are tight on gel cassette

    b. Tighten electrodes

    c. Put in gel cassette

    d. Put in clamp and dummy gel (in slot closest to the clamp)

    e. Get running buffer and bucket of ice

    f. Get large tray and fill bottom with ice; place mini cell on top of ice

    g. Take out a gel and cut open the package carefully

    h. Remove the white strip on the bottom of the gel

    i. Take out the comb vertically and rinse the wells with diHO; flick 2

    out the water in the sink

    j. Place the gel into cell (in slot furthest from clamp) and clamp it

    shut

    i. Make sure everything is secure and tight

    3. immediately fill middle chamber with running buffer all the way to the top

    (enough to cover the wells of the gel)

    4. wait a minute to make sure the chamber isn’t leaking

    5. microcentrifuge samples

    6. get Novex MW marker (keep on ice) & prep

    a. vortex MW marker, make 1:1 ratio of LDS sample buffer : Novex

    MW marker

    b. vortex & microcentrifuge when done

    7. fill rest of chamber with running buffer up to where the white tape strip was

    on the gel

    8. make a loading template of the wells in your notebook

    SKG 8/4/10

    Gel Electrophoresis & Western Blot Protocol

    9. load wells with GEL LOADING TIPS

    a. *PRIME EACH TIP* with whatever reagent you’re loading

    b. lower tip into well slowly, holding the tip against the plastic side of

    the gel casing

    c. go to the bottom of the well, but do not touch the gel with the tip

    d. expel SLOWLY and let the solution reach the bottom of the well

    e. load empty wells with 1x LDS sample buffer

    f. does your loading match your template?

    g. Tip: mark an X on each well in your notebook template to indicate

    which wells you’ve loaded already

    10. put lid on cell and surround the cell with ice

    11. plug into power supply

    a. plug cell in first!

    b. Plug in power supply, then turn on switch in back

    12. Power Supply Commands: select “NuPAGE Gel” ; 1 hr 30 min ; 150V

    ; Start

    a. check for bubbles to make sure it’s running properly

    13. come back with 30-45 minutes left to prep for Blotting:

    14. get the following out:

    a. in the hood, fill the “Methanol Only” dish with about ? in. of

    methanol put the lid on after until you’re ready to use it

    b. remove your cold transfer buffer from the fridge and fill the

    “transfer buffer only” dish with 1-2 in. of transfer buffer

    c. get out 4 sponge pads, 2 filter papers, and 1 PVDF or

    nitrocellulose membrane per gel

    d. get your gel knife, angled tweezers, and a clean tray ready

    e. lay out paper towels wherever you’re working to keep your tools

    clean. Don’t put tweezers or knife on dirty surfaces; you’ll

    contaminate your membrane

    15. place your sponge pads in the “transfer buffer only” tray and submerge

    them. Pound the sponge pads with the handle of the gel knife to remove

    bubbles. When done, place your filter papers in the same tray and

    submerge them too. Do not pound them.

    16. with 10 minutes left, place your membrane in the methanol and place it on

    the rocker with the lid on. set a timer for 10 minutes

    17. now your gel should be done. Turn off the power supply, THEN remove

    the lid to the cell.

    SKG 8/4/10

    Gel Electrophoresis & Western Blot Protocol

    18. Begin making your sandwich:

    a. Place the larger of the white plastic sandwich sides down (with

    the sides, and assemble as follows (from bottom up):

     Sponge pad Sponge pad

    Filter Paper

    Membrane

    Gel

     Filter Paper

     Sponge pad

     Sponge pad

    b. Removing the gel: (Be very GENTLE. It rips easily)

    i. Only remove the gel when you’re ready to place it on (after

    you’ve layered the pads and filter paper.

    ii. Remove the gel from the chamber and dump the running

    buffer into the “recycled running buffer” container & put it

    back in the fridge

    iii. Break the seals on the gel with the gel knife and remove one

    side carefully

    iv. Cut the wells off the top of the gel GENTLY and dispose of

    them in the “Polyacrylamide waste” container in the hood

    v. Cut the bottom (about ? in.) of the gel off, right below the

    blue bands that identify the bottom of the run.

    vi. Dip your hand into the transfer buffer tray and sprinkle some

    transfer buffer on the gel and gel knife

    vii. Use the gel knife to lift up the corner of the gel and use your

    hands (wet with transfer buffer) to lift the gel off of the plastic

    viii. Immediately put gel into the sandwich and align it correctly,

    wetting it continuously with transfer buffer

    b. Make sure the membrane covers the gel completely and it is

    straight. The proteins will transfer in whatever orientation you

    align them in, so be sure to make everything straight

    c. Use the gel knife to smooth out any bubbles between the

    membrane and gel

    19. after completing the sandwich, pour some more transfer buffer on the top and place the top side of the cassette on the sandwich, but do not squeeze it down (once it’s squeezed, you cant let go until you put it in the chamber or else you’ll let in air bubbles)

    20. rinse the cell with di water and place in the lock

    21. squeeze the sandwich closed, place it in the cell, and lock the cell

    22. fill the sandwich to the top with transfer buffer (but leave about a centimeter on the top or you’ll disrupt the current)

    SKG 8/4/10

    Gel Electrophoresis & Western Blot Protocol

    23. fill the back of the cell (around the lock) with ice

    24. fill the rest of the cell with cold water (from your melted ice) about 1/3 of

    the way

    25. place the lid on the cell and turn on the power supply 26. “NuPAGE Blot” ; 2 hours, 100V ; “start”

    27. anytime before your blot is done, make your 5% milk solution

    a. ex. 5g of nonfat dry milk, 100mL TBS-Tween

    b. weigh out milk in “Western blot milk” beaker and add in TBS-

    Tween. Place in stir bar and leave on stirrer until you need it.

    c. You’ll need 10mL milk per strip

    28. when blot is done:

    a. disassemble cell and wash all parts with di water and let dry in

    rack

    b. dispose of gel in “polyacrylamide waste” beaker

    c. throw away filter papers, save sponge pads and rinse in di water

    d. lay membrane on “membrane cutting board” board

    e. wet membrane with some transfer buffer or TBS-T

    29. cut membrane according to proteins you’re probing for

    a. use a clean razor

    b. use a straight edge (lid of a 24-well plate)

    c. cut straight across! Use a ruler to measure both sides if

    necessary

    30. place strips into clean strip box

    31. label each compartment with colored tape on the lid with protein, animal it

    came from, name, date

    32. if not blocking immediately, put some TBS-T in the box to keep strips wet 33. block membranes for 30 minutes with 10mL milk per strip on rocker 34. remove milk and wash twice with TBS-T

    35. dump in an entire conical of 1 Antibody solution for each well. Pay

    attention to the labels to make sure everything matches up. Put conicals

    back in fridge so you can return the antibody solution tomorrow. 36. take box to Biotech Core cold room and place on rocker (tape box to

    secure it to the rocker)

    37. incubate in 1 Ab overnight

    DAY 2 Developing Western Blots

38. Retrieve strips from cold room

    39. pipette 1 Ab back into respective conicals, paying attention to labels. Put

    1 Ab solutions back in the 4C

    40. wash strips with TBS-T for 30 min, changing TBS-T at 5 min. increments

    a. while washing strips, make a 5% milk-TBS-T solution just as in

    Day 1 (you’ll need 10mL per strip again)

    41. when done washing strips, pour out TBS-T and put 10mL 5% milk into

    each slot

    SKG 8/4/10

    Gel Electrophoresis & Western Blot Protocol

    42. add 2.5 ;L of 2 Antibody to each slot. Be sure to pay attention to the

    animals! This is why you labeled the strip with the 1 Ab animal. Use the

    2 Ab that is anti-“whatever you used for 1 Ab”

    a. pipette the antibody up and down in the milk solution to wash out

    all of the 2 Ab

    43. incubate strips with 2 Ab on rocker for 1 hour

    44. wash strips for 30 min with TBS-T at 5 min increments

    45. Developing

    a. Make a note of which ECL you’ll use (regular or Plus). Plus is a

    more sensitive developer and should be used for proteins that are

    harder to get bands out of. If you develop with regular and don’t

    see anything, you can wash off the ECL with TBS-T for 10 min

    and repeat with ECL Plus

    b. Use Developing kit (here is the protocol for GE kit at the VA) all

    kits are stored in fridge

    i. ECL regular: let reagents reach room temp. make 1:1 Ratio

    of Reagent 1 to Reagent 2

    ii. ECL Plus: 4mL of Reagent A, 100;L of Reagent B

    c. Have your autoradiography box, film, and sheet protectors ready d. Take strips out of box one at a time and drag them across the

    clean surface you’re using (ex: a cutting board) to dry them.

    Touch the edge of the strip to a paper towel to get off excess

    TBS-T

    e. Do this for all strips and align them on the sheet f. Pour ECL solution over strips and wait about 5 min g. Dry strips again by dragging and touching them to a paper towel h. Place strips in sheet protector and align them in the correct order

    to “re-piece” the original gel

    i. Close the sheet protector and smooth out the bubbles by wiping

    the top with a paper towel

    j. Tape the sheet protector into the autoradiography box. Again,

    make sure the strips are parallel to the bottom of the box. If it’s

    crooked, your bands will come out crooked.

    k. Take autoradiography box, timer, film, notebook, and marker to

    darkroom

    l. With the LIGHTS OFF, take out a sheet of film and bend the top

    left corner to indicate which way is top left

    m. Place film in the autoradiography box, lock it, and set timer for

    how ever long you want to expose

    n. Take out film and place into developer (still with lights off) o. Repeat with a film for each development time you want

    i. Usually, it’s good to start with 30 seconds and see how it

    comes out. Then gauge the other times (1 min, 2 min) if it

    comes out too dark, try going lower (15 sec, 5 sec. etc.)

    ii. The developer will beep about a minute after you put in a

    film. This indicates that you can put in another film.

    SKG 8/4/10

    Gel Electrophoresis & Western Blot Protocol

    iii. Get a good range of times (30 sec, 1 min, 2 min, or 2 min, 3

    min, 5 min, etc)

    p. The light can be on when your film is in the developer (just make

    sure no undeveloped film is out whenever the lights are on) 46. when back in the lab, remove the strips from the sheet protector and place

    back into strip box with fresh TBS-T

    47. pick the best films and scan them into the computer. Email Dr. Nogal and

    yourself the pictures or put on flashdrive.

    48. bring strips and films back to lab. Put films in “Western Blot Films” binder.

    Download the scanned images and put them in the “WB Images from VA”

    folder on the lab computer (login under username “Nekeisha”)

    49. if probing for more proteins, you’ll need to strip the membranes and probe

    with the corresponding 1 Ab

    a. empty TBS-T from strip box

    b. add ~6mL stripping solution per strip and rock for 30 min. max

    c. dump off stripping solution and wash strips for 30 min at 5 min

    increments. Make 5% milk solution again.

    d. Block with 5% milk solution for 30 min

    e. Re-label strip box

    f. Add in 1 Ab for the different proteins you’re probing for and save

    the conicals

    g. Incubate overnight in cold room and repeat DAY 2 procedure to

    develop again (starting with returning 1 Ab, washing for 30 min,

    incubating in 2 Ab solution, etc)

    50. throw strips away when completely done with procedure. Rinse all

    materials with di water

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