Protocol: Western Blot



Michael’s Protocol: Western Blots

1) Protein Extraction

a) [Cell pellet and protein extract should be kept on ice at all times or done in a cold room]

b) Thaw cell pellets on ice

c) Mix lysis buffer (60 ul per sample)

i) 594x MPER Buffer (Thermo) (Room Temperature) (Sudeshna’s shelf)

ii) 6x Protease Inhibitor (100x) (-20C) (left –20C fridge top drawer in “Protease Inhibitor” box) (minimize freeze and thaw cycle)

d) Add 60ul MPER Buffer to pellet and pipet up and down

e) Incubate pellet on ice for 10 minutes

f) Spin extract in a centrifuge in the cold room at 14x103 rpm (or max speed) for 10 minutes, cell debris, DNA, and other materials will form a pellet at the bottom of the tube allowing the proteins to be suspended in the supernatent (extract must be ice cold before loading into centrifuge)

g) Transfer supernatent, about 100 ul

i) If the supernatent is murky, more lysis buffer can be added and protocol repeated

2) Protein Concentration Determination (BCA Assay) (Sudeshna’s 3rd drawer)

a) Protein concentration is measured using the BCA Assay, other methods may be used

b) BSA standards should be made prior to protein determination

i) 1mg/1mL MPER Buffer – 5mg/1mL MPER Buffer (BSA is stored as a solid at 4C in PCR fridge)

c) Mix Reagent “A” “B” (200ul per sample): (Will turn green)

i) 5000x Reagent A

ii) 100x Reagent B

d) Using a 96-well culture dish, add 200ul of AB solution in each well

i) 1 mock reaction, 1 MPER buffer only, 5 standards, 1 for each sample

ii) Add 2ul of Buffer, 2ul of each standard, and 1ul of each sample

e) Mix well

f) Incubate at 37C (E coli. Incubator) for 30 minutes (solutions will turn pink/purple)

g) Take out plate and use Envision Manager to measure absorbance at 560nm

i) Use Protocol “Copy of Copy of BCA” in RHBaker

ii) Use “Blank” for Reagent and mock samples

iii) “Standard” for standards

iv) “Unk” for unknowns

v) Use least squares to linearize standards and apply unknowns to standard curve

3) Protein Loading Preparation (for reducing and denatured sample) (Be sure to prepare positive control as well!!!)

a) Prepare Laemmelli buffer (5x) (Room Temperature)

b) Mix:

i) 95x Laemmelli Buffer (5x)

ii) 5x DTT (1 M) (-20C) (left -20C top shelf in blue eppendorf rack)

c) Aliquot one 30ug sample with 6ul mixed Laemmelli Buffer and dilute to a final volume of 30ul in MPER buffer (Volume must be exact to ensure actin bands are identical)

d) Mix samples very well by flicking

e) Heat aliquot at 95C Aluminium heat block for 5 minutes, alternatively protein can be heated at 65C for 10 minutes

f) Mix samples by flicking

g) Freeze denatured protein sample at –20C until ready to load on gel

h) Freeze remaining protein extract in aliquots at –80C, aliquots must be small enough to minimize freeze and thaw cycles to at most two (alternatively, all the protein sample can be denatured at one time)

4) Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (ensure preparations for transfer are finished before the gel is finished running)

a) Mix Running Buffer: (Running and transfer buffers can be reused for a maximum of three times)

i) 100x Tris/Glycine/Sodium Dodecyl Sulfate (10x)

ii) 900x ddH20

b) Spin down denatured protein aliquot to ensure volume and concentration are precise

c) Obtain a 7.5 ug protein aliquot from the protein sample (7.5ul) and dilute in 7.5ul of 50% glycerol and 50% MPER Buffer

d) Obtain a 4-20% polyacrylamide gel and cut along bottom of gel, peeling plastic to expose gel

e) Place gasket in gel running box and place gel in box making sure that there are no leaks

f) Place in running box

g) Fill gel running box with running buffer to ensure that there are no leaks

h) Wash wells of gel by pippeting running buffer inside of well

i) Then fill running buffer up to appropriate line on running box

j) Load 15ul positive control (786 VHL- for Cyclin D1) and 10ul of PageRuler Plus (black) (-20 C Left fridge) in gel, alternate order of loading to differentiate gels

i) Ensure that there are no bubbles

ii) Ensure that protein is loaded very slowly

k) Load 15ul of each sample, do as quickly as possible in order to minimize diffusion of protein

l) Run gel at 100-120V for about 1 hour, when bromophenol blue reaches bottom of gel and leaks out

5) Transfer

a) Must be done immediately and quickly after gel is finished running, keep gels that you are not working on in the running buffer

b) Never directly touch membrane, always use tweezers!

c) Mix transfer buffer: (Ensure buffer is mixed before gel is finished running) (Use more methanol for smaller proteins and less methanol and more SDS for larger proteins)

i) 1x Transfer Buffer (10x)

ii) 7x ddH2O

iii) 2x Methanol

d) Cut membrane and blot paper to size of gel

e) Incubate PVDF in cold methanol for 1-2 minutes, then 3-5 minutes in ice cold Transfer Buffer (must be done before gel is finished running)

f) When gel is done running, release gel by cutting along white line with razor

g) Place gel on dry blot paper and put in transfer buffer immediately to prevent shrinking and tearing

h) Cut wells and bottom of gel off to ensure gel is flat

i) Make sandwich of gel sponge/blot paper/blot paper/gel/membrane/blot paper/blot paper/sponge making sure that gel is next to black side of cassette (Make sure to make sandwich under transfer buffer in order to prevent the formation of bubbles

j) Roll sandwich with 15mL tube to remove bubbles and close cassette tightly under transfer buffer

k) Place cassette in transfer box, black to black, and place in running box

l) Run in cold room for 75 minutes at 100V

6) Blocking

a) Mix TBST Buffer:

i) 100x TBS Buffer (10x)

ii) 899x ddH20

iii) 1x Tween20

b) Mix 5% nonfat milk:

i) 5 g nonfat milk

ii) 100 mL TBST

c) Remove cassette from transfer box

d) Remove membrane carefully and place in clear pipet box

e) Add enough 5% nonfat milk to cover membrane, approximately 10mL in a 200ul pipet box

f) Incubate at room temperature with agitation for 1 hour

7) Primary Antibody

a) Cut plastic cover to appropriate size to fit one membrane

b) Place membrane inside of plastic cover, make sure at least 3 sides of the cover are open in order to easily place membrane in

c) Seal 3 sides of cover and add 4mL of 5% milk, ensure that milk does not sediment

d) Gently squeeze out all bubbles and check to see if there are leaks

e) Add antibody at appropriate dilution

i) 1:10000 for actin (right –20C antibody box)

ii) 1:1000 for Cyclin D1 (4C human antibody box)

f) Incubate overnight at 4C for not more than 24 hours

8) Secondary Antibody

a) Pipet out primary antibody and save at 4C for at most 3 days

b) Wash membrane three times with TBST with agitation, 10 minutes for each wash, longer washes may decrease background

c) Dilute secondary antibody in 10 mL of 5% milk (1:10000 dilution)

d) Incubate membrane in 10mL of diluted antibody at room temperature for 90-120 minutes with agitation

e) After incubation, dispose milk and wash with TBST at least four times

9) Film Development

a) Mix equal volumes of reagent A and B of Electrochemical luminescence kit (4C antibody fridge)

b) Immediately apply to membrane, approximately 1mL per membrane

c) Dry membrane with kimwipe or let it drip dry in the air

d) Place membrane in pre cut folder cover and tape in place in film exposure box

e) Proceed immediately to dark room with film, timer, and membranes

f) Never expose film to light!!!

g) Place film on one side of box, making sure that it is on the edge, and close box for 5 seconds

h) Take film out and flip over to repeat at 30 seconds

i) Expose film in detector to gauge how long to expose film in order to achieve nice bands within a linear range

j) When finished, ensure that film is in box before turning on the lights

Buffer Components (from Sudeshna):

Lammelli Buffer (5X): 4 ml 1.5M Tris-HCl Ph= 6.8, 10 ml glycerol, 2g SDS, Bromophenol Blue. Just before preparing samples add DTT (stock 1M) to a final concentration of 50mM

Running Buffer: Use either 10X Tris/Glycine/SDS Buffer from BioRad (Cat#161-0732), or prepare 10X Running buffer using the following recipe:

Running Buffer (10 X, 1 liter): 30.3 g Tris Base, 14.42 g glycine, 10 grams SDS, adjust pH to 8.3 with conc HCl.

Wet Transfer Buffer: (10X, 1 liter): 30.3 g Tris Base, 144.1g.

Wash Buffer (10X, 1 liter): 24.23 g Trizma.HCl, 80.06g NaCl.

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