Cells were lysed in cell lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA, 0.5% (v/v) sodium deoxycholate, 1% (v/v) NP-40, pH 8) with protease and phosphatase inhibitor for 30 min. The lysates were centrifuged at 14,000 × g for 15 min at 4°C, and the supernatant protein was quantified using BCA assay. Total protein was normalized, mixed with 1x SDS-PAGE loading buffer, denatured at 95°C for 5 min and resolved on an SDS-polyacrylamide gel, and transferred to PVDF membrane. For dot blots, conditioned media was blotted on a nitrocellulose membrane without boiling. Membranes were blocked with 5% bovine serum albumin (Sigma). Blots were probed with primary antibodies to LAMP1 (abcam ab108597), GCase (abcam ab55080), pT73-Rab10 (abcam ab230261), Rab10 (cell signaling 8127S), pT72-Rab8a (abcam ab230260), Rab8a (abcam ab188574), pS935-LRRK2 (abcam 133450), LRRK2 (clone, 8629). αSyn oligomer (abcam ab209538). Secondary antibodies conjugated to horseradish peroxidase were used for detection using autoradiography.
Ab108597
Manufactured by Abcam
Ab108597 is a primary antibody that can be used for the detection of a specific target protein. The core function of this product is to provide researchers with a tool for analysis and identification of the target protein within biological samples.
Automatically generated - may contain errors
2 protocols using ab108597
1
Immunoblot Analysis of Lysosomal Markers
2
Immunoblotting for Protein Expression
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Cells were lysed in cell lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA, 0.5% (v/v) sodium deoxycholate, 1% (v/v) NP-40, pH 8) with protease and phosphatase inhibitors for 30 min. Lysates were centrifuged at 14,000g for 15 min at 4°C, and supernatants were quantified using the BCA assay. Samples were prepared in 1x SDS-PAGE loading buffer, denatured at 65°C for 10 min, resolved on an SDS-polyacrylamide gel, and transferred to PVDF membrane. Membranes were blocked with 5% BSA (Sigma) prior to probing with primary antibodies against LAMP1 (abcam ab108597), Rab10 (abcam ab237703), Rab8a (Cell Signalling 6975), αSyn oligomer (abcam ab209538), total αSyn (MABN1817), pTau (Ser202, Thr205) (Thermo; MN1020), and total Tau (13–6400). Secondary antibodies conjugated to horseradish peroxidase were used for detection using autoradiography.
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