Cultured cells were lysed in 50 mM Tris, pH 7.5; 150 mM NaCl; 1% (v/v) Triton X-100; 0.5% (w/v) sodium deoxycholate; 0.1% (w/v) sodium dodecyl sulfate; 5 μg/mL aprotinin, 1 μg/mL leupeptin, 1 μg/mL pepstatin, and 1 mM Pefabloc. After clarification of the lysates by centrifugation, protein concentrations were determined using the Micro BCA Protein Assay Kit. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes by semidry-blotting. Antibodies were purchased from the following providers: Merck Millipore (α-actin mAb1501, dilution 1:5000); Cell Signaling Technology (α-tubulin clone DM1A, dilution 1:2000; α-GAPDH clone 14C10, dilution 1:1000); Santa Cruz Biotechnology (α-PGP clone E10, dilution 1:75). Rabbit polyclonal α-PGP antibodies (employed at 1:1000 dilution) were generated by Charles River using purified, untagged full-length murine PGPD34N as an immunogen in New Zealand White rabbits, and antibodies were purified by affinity chromatography11 (link).
α tubulin clone dm1a
Manufactured by Santa Cruz Biotechnology
α-tubulin (clone DM1A) is a primary antibody that specifically recognizes the α-tubulin subunit of the tubulin heterodimer, a key structural component of microtubules. This antibody is commonly used in various research applications to detect and visualize the cytoskeletal network within cells.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by GE Healthcare (2 mentions)
Ripa lysis buffer, by Merck Group (2 mentions)
Tween 20, by Merck Group (2 mentions)
Restore western blot stripping buffer, by Thermo Fisher Scientific (2 mentions)
Thepierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bovinealbumin fraction 5, by MP Biomedicals (2 mentions)
Phospho p44 p42 mapk erk1 2 thr202 thy204, by Cell Signaling Technology (2 mentions)
Pax8 clone pax8r1, by Abcam (2 mentions)
E cadherin clone 24e10, by Cell Signaling Technology (2 mentions)
β actin clone d6a8, by Cell Signaling Technology (2 mentions)
Complete mini edta free, by Roche (2 mentions)
3 protocols using α tubulin clone dm1a
1
Immunoblotting of Cell Lysates
2
Protein Expression Analysis in Mouse Thyroid Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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SPTL and mouse thyroid follicular cells were lysed with RIPA Lysis Buffer
(MilliporeSigma) with proteinase inhibitor cocktail, cOmplete mini EDTA-Free (Roche). The
Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) was used for measuring the protein
concentration of lysates. Samples were subjected to SDS-PAGE and transferred to PVDF
membranes (GE Healthcare). Tris-buffered saline (TBS) buffer containing 5% bovine
albumin fraction V (MP Biomedicals) and 0.1% Tween-20 (Sigma-Aldrich) was used for
blocking. Stripping of membrane was performed using Restore Western Blot Stripping Buffer
(Thermo Fisher Scientific). Antibodies used were those against AKT (#9272, Cell
Signaling Technology), phospho-p44/p42 MAPK (ERK1/2) (Thr202/Thy204) (#9101, Cell
Signaling Technology), ERK1+ERK2 (ab17942, Abcam), PAX8 (clone PAX8R1, ab53490, Abcam),
E-cadherin (clone 24E10, Cell Signaling Technology), α-tubulin (clone DM1A,
sc-32293, Santa Cruz), and β-actin (clone D6A8, #8457, Cell Signaling
Technology). Anti-NKX2-1 and anti-phospho-AKT (Ser473) antibodies were the same as those
used for immunohistochemistry/immunofluorescence.
(MilliporeSigma) with proteinase inhibitor cocktail, cOmplete mini EDTA-Free (Roche). The
Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) was used for measuring the protein
concentration of lysates. Samples were subjected to SDS-PAGE and transferred to PVDF
membranes (GE Healthcare). Tris-buffered saline (TBS) buffer containing 5% bovine
albumin fraction V (MP Biomedicals) and 0.1% Tween-20 (Sigma-Aldrich) was used for
blocking. Stripping of membrane was performed using Restore Western Blot Stripping Buffer
(Thermo Fisher Scientific). Antibodies used were those against AKT (#9272, Cell
Signaling Technology), phospho-p44/p42 MAPK (ERK1/2) (Thr202/Thy204) (#9101, Cell
Signaling Technology), ERK1+ERK2 (ab17942, Abcam), PAX8 (clone PAX8R1, ab53490, Abcam),
E-cadherin (clone 24E10, Cell Signaling Technology), α-tubulin (clone DM1A,
sc-32293, Santa Cruz), and β-actin (clone D6A8, #8457, Cell Signaling
Technology). Anti-NKX2-1 and anti-phospho-AKT (Ser473) antibodies were the same as those
used for immunohistochemistry/immunofluorescence.
3
Protein Expression Analysis in Mouse Thyroid Cells
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