If high background is observed, replace milk with 5% (w/v) BSA in TBS-T for blocking and antibody dilution as phosphor-specific antibody detects casein in the milk.Add phosphatase inhibitors and keep samples on ice at all times to preserve the phosphorylated state of the proteins.The blocking and incubation agents used were not freshly prepared or were too dilute.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.

Immunoblotting vs western blot