Western Blot protocol
· Prepare ice bucket to place cell culture dishes. Pre-cool table top centrifuge.
· Aspirate media,
· Wash with ice cold PBS
· Aspirate PBS
· Add Lysis Buffer to cells, scrape cells and transfer to pre-cooled Eppendorf tube
· Lyse on ice for about 10 minutes
· Spin at 13,000 rpm in table-top centrifuge for 10 min
· Transfer supernatant to a fresh pre-cooled Eppendorf tube discard the pellet.
· Aliquot 10 to 20µl of lysate for protein determination.
· Store protein at –20 or -80 for long term storage. Avoid multiple freez and thaw.
· Always store protein lysates at -80°C. Always thaw protein lysate on ice.
Sample preparation:
· Make sure that you added the ß-Mercapto to the 4x SLB.
· Always work on ice.
· Prepare 20µg of protein lysate per lane in a final volume of around 25 µl per lane. When working with TENTE/4x SLB Buffer:
· Xµl Protein lysate (20µg) + 6,25 µl 4x SLB + (25-6.25-X)µl 1x TENTE Buffer.
· Heat at 95°C for 5 min. then put on ice.
· This sample can be stored at -20 and
Casting Gels
· Clean plats first with water, then with EthOH. Match and makesure that the ottoms of the plates are aligned in a flat surface (flush). Clip into a green Biorad holder. Test leakiness by putting water and then soak water with filter paper.
· Put the comb and mark just below the comb for the level of running gel.
· Mix and poir separating gel (about 8 ml required for one 1.5mm width gel). Pour up the running gel mark.
· Seal the top of the gel by GENTLY pipetting a thin layer of isopropanol/EtOH on top. Keep the remaining acrylamide in a falcon tube with the lid closed, this will tell you when the gel has polymerized.
· Mix and pour stacking gel (about 5 ml per 1.5mm gel).
· Insert a clean comb (make sure it is the same with as the glass plates). Consider wearing goggles, definitely wear gloves because acrylamide will spill/squirt.
· After stacking gel has polymerized, remove combs.
· Put gel plates into Biorad gel-running apparatus. Make sure smaller gel plate of gel sandwich is facing in.
SDS PAGE gel
Loading and Running Gel· Pour 1x running buffer till it totally fills up the chamber between the gels and fills of the outer main chamber.
· Load gel: load desired amount of protein (equal amount of protein ~ 20 µg) and protein marker
· Run stacking gel around 50V, then increase voltage to 100-120V for about an hour until the “blue” runs out.
Please follow this youtube video
Transferring: WET transfer
· Make 1x transfer buffer (1.5 l, can be reused twice)
· Take out gel sandwich, open, remove and discard stacking gel.
· Inculate the gel in transfer buffer in a large, flat glass dish.
· Cut a pvdf/nitrocellulose membrane approx. the same size as the gel. Submerge in 100% Methnol to hydrate for 1 to 2 min. Then move to transfer buffer. Cut at least 4 sheets of whatman paper slightly bigger than membrane.
· Set up transfer sandwich in transfer buffer.
Bottom to top:
· 2 sheets of whatman paper. Gel, membrane, 2 sheets whatman. Roll out bubbles with 25ml pipette or a roller. Add 1-2 wet sponges to each side of sandwich, then place on top of black side of pastic sandwich holder (gel should still be below membrane, closest to the black plastic!).
· Close sandwich holders and insert black side toward black side of the transfer apparatus.
· Put the whole thing in the buffer chamber, add an ice block.
· Fill with transfer buffer to top, place in 4°C room on magnetic stirrer. Ad stirring bar. Run 1 -1.5h at 100V. Check that transfer is done by opening sandwich an peeling back the side of the gel containing the prestained protein marker. If the mrker has totally transferred to the membrane, the transfer is done (If not, but the sandwich back in a little longer).
· Open sandwich, Tilt membrane till you can see the shiny lanes of protein. Also cut off the top right corner of the membrane for orientation.
· Put membrane face up in flat bottom plastic box. Add 5% milk (or BSA, depending on antibody) diluted in TBST. Block at RT 60 min. On a rocker.
· Add primary antibody diluted in 5% milk in TBST. Place in cold room on rocker overnight (with lid on box, orient so that milk is rocking from top to bottom of the membrane, not from side to side. Do not use a box that requires more than 3.5-4ml of primary antibody solution. If you do not have this kind of box available, stick membrane into 50ml falcon tube (protein side facing inward), add antibody solution, and incubate o/n on “rolling tubes”.
· Next day take out the membrane and wash 5 times for 5 min each with TBST
· Incubate with appropriate secondary antibody for an hour at room temperature
· wash 5 times for 5 min each with TBST
· Develop the blot with ECl and take a nice image
· 2 sheets of whatman paper. Gel, membrane, 2 sheets whatman. Roll out bubbles with 25ml pipette or a roller. Add 1-2 wet sponges to each side of sandwich, then place on top of black side of pastic sandwich holder (gel should still be below membrane, closest to the black plastic!).
· Close sandwich holders and insert black side toward black side of the transfer apparatus.
· Put the whole thing in the buffer chamber, add an ice block.
· Fill with transfer buffer to top, place in 4°C room on magnetic stirrer. Ad stirring bar. Run 1 -1.5h at 100V. Check that transfer is done by opening sandwich an peeling back the side of the gel containing the prestained protein marker. If the mrker has totally transferred to the membrane, the transfer is done (If not, but the sandwich back in a little longer).
· Open sandwich, Tilt membrane till you can see the shiny lanes of protein. Also cut off the top right corner of the membrane for orientation.
· Put membrane face up in flat bottom plastic box. Add 5% milk (or BSA, depending on antibody) diluted in TBST. Block at RT 60 min. On a rocker.
· Add primary antibody diluted in 5% milk in TBST. Place in cold room on rocker overnight (with lid on box, orient so that milk is rocking from top to bottom of the membrane, not from side to side. Do not use a box that requires more than 3.5-4ml of primary antibody solution. If you do not have this kind of box available, stick membrane into 50ml falcon tube (protein side facing inward), add antibody solution, and incubate o/n on “rolling tubes”.
· Next day take out the membrane and wash 5 times for 5 min each with TBST
· Incubate with appropriate secondary antibody for an hour at room temperature
· wash 5 times for 5 min each with TBST
· Develop the blot with ECl and take a nice image
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