teragasil.blogg.se - Western blot protocol

Post-antibody washes may not have been performed for a sufficient period of time or were not performed in a high enough volume.Contamination can be transferred to the blots from electrophoresis and related equipment used in blot preparation. Transfer buffers may have become contaminated.Exposures can vary from 5 seconds to 60 minutes. Place the wrapped blots, protein side up, in an X-ray film cassette and expose to x-ray film.Drain off the excess detection reagent, wrap up the blots, and gently smooth out any air bubbles.The final volume required is 0.125ml/cm 2. Incubate membrane (protein side up) with 10ml of ECL (enhanced chemiluminescence substrate) for 1-2 minutes.Wash 4 times for 10 minutes each with TBS containing 0.1% Tween-20 and once for 2 minutes with PBS.Incubate the membrane for 30 minutes at room temperature with horseradish peroxidase (HRP)- conjugated secondary antibody, diluted to 1:1000 - 1:5000 in 5% nonfat dry milk/ TBS-T.Wash three times for 5 minutes each with Wash Buffer (TBS containing 0.1% Tween-20).Place the membrane in the primary antibody solution and incubate for 2 hours at room temperature, or overnight at 4☌ with agitation. Dilute the primary antibody to the recommended concentration/dilution in 5% nonfat dry milk/TBS-T.Incubate the blot for 1 hour at room temperature, or overnight at 4☌ with agitation.Remove the blotted membrane from the transfer apparatus and immediately place in blocking buffer consisting of 5% nonfat dry milk/TBS-T**.After boiling, continue as normal to the membrane blocking step of the protocol. Immediately after transferring the gel onto the membrane, submerge the membrane in boiling PBS for 5 minutes. Transfer the proteins to a nitrocellulose or PVDF membrane with variable power settings according to the manufacturer’s instructions.įor Amyloid Beta Detection, Boiling Method: Set gel running conditions according to the manufacturer’s instructions.*Guidelines for choosing gel percentages are based on protein size to be detected: 4-5% gel, > 200 kD 7.5% gel, 120-200 kD 8-10% gel, 40-120 kD 13% gel, 15-40 kD 15% gel, < 20 kD. Load up to 40µl of sample to each well of a 1.5mm thick gel*.

Remove 20µl of supernatant and mix with 20µl of 2x sample buffer.

Transfer the supernatant to a new tube and discard the pellet.

Lyse the cell pellet with 100µl of lysis buffer on ice for 30 min (For 1 X 10 6 cells, lyse with 100µl of lysis buffer).

Place cells in a microcentrifuge tube and centrifuge to collect the cell pellet.

10X TBS-T (Tris-buffered saline containing Tween-20): Dissolve 80g of NaCl, 2g of KCl, 30g of Tris base and 10ml Tween-20 in 800ml of distilled H 2O.

Blotting Membrane: Nitrocellulose or PVDF membrane.

Alternate Primary and Secondary Antibody Dilution Buffer: 1X TBS-T with 4% Bovine Serum Albumin (BSA).

Alternate Blocking Buffer: 1X TBS-T with 4% Bovine Serum Albumin (BSA).

Primary and Secondary Antibody Dilution Buffer: 1X TBS-T with 5% nonfat dry milk.

Blocking Buffer: 1X TBS-T with 5% nonfat dry milk.

Transfer Buffer: 3.0g Tris base, 14.4g Glycine, 200ml Methanol.

10X SDS Running Buffer: Dissolve 144g of Glycine, 30g of Tris base and 10g SDS in 800ml of distilled H 2O.1X Cell Lysis Buffer: 20 mM Tris-HCl (pH 7.5), 150mM NaCl, 1% NP-40, 2 mM EDTA, 1µg/ml leupeptin, 1µg/ml aprotinin, 1 mM Na 3PO 4, 1 mM PMSF, 5 mM NaF, 3 mM Na 4P 2O 4.