Anti-Smad4/DPC4 Antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Rabbit (0.1 µg/mL)

Protocol tips
-Rabbit (0.1 µg/mL)

Publication protocol

Adipose tissue samples were homogenized (FastPrep®‐24, MP Biomedicals, Santa Ana, CA) in two volumes of ice‐cold cell lysis buffer (Invitrogen, Burlington, ON) supplemented with protease inhibitor cocktail (as per the manufacturer's instructions; Sigma‐Aldrich, Oakville, ON, Canada) and 0.05% of phenylmethylsulfonyl fluoride (BioShop, Burlington, ON, Canada). Homogenized samples were centrifuged for 10 min at 1,500g at 4°C. Lipids were removed, the infranatant was collected, and protein concentration was determined with a bicinchoninic acid assay (28) (ThermoScientific, Rockford, IL). Western Blotting was performed as described previously by our laboratory (29-32). Briefly, membranes were incubated in primary antibodies diluted in TBST/5% nonfat dry milk (COX4 [CAT#:MS407], CORE1 [CAT#: MS303]: MitoSciences, Eugene, OR; β‐actin [CAT#: ab8227]: Abcam, Toronto, ON, Canada; adipose tissue triglyceride lipase [ATGL] [CAT#:2138], p‐SMAD2 [ser465/467] [CAT#:3101]: Cell Signaling, Danvers, MA, USA) or TBST/5% bovine serum albumin (phosphoenolpyruvate carboxykinase [PEPCK] [CAT#:10004943]: Cayman Chemicals, Ann Arbour, MI; ERK1/2 [CAT#:4695], p‐ERK1/2 [thr202/tyr204] [CAT#:9101], hormone‐sensitive lipase [HSL] [CAT#:4107], SMAD2 [CAT#: 5339], SMAD3 [CAT#: 9523], SMAD5 [CAT#:9517], p‐SMAD1 [ser463/465] /5[ser463/465] /8[ser426/428] [CAT#: 9511], p‐SMAD3 [CAT#: 9520], JNK [CAT#:9252], p‐JNK [thr183/tyr185] [CAT#:4671], TGF‐β Receptor I [CAT#:3712], TGF‐β Receptor II [CAT#:11888]: Cell Signaling; SMAD1 [CAT#:sc‐81378], SMAD8 [CAT#:sc‐11393]: Santa Cruz Biotechnology, Dallas, TX; SMAD4 [CAT#:ABE21]: Millipore, Billerica, MA; SMAD6 [CAT#:PA1‐410216], SMAD7 [CAT#:PA1‐41506]: ThermoScientific; SMURF2 [CAT#:ab53316], Cytochrome C [CAT#:ab110325], DGAT1 [CAT#:ab54037], GPAT [CAT#:ab69990], α‐tubulin [CAT#: ab7291]: Abcam; SMAD Anchor for Receptor Activation [SARA] [CAT#:GTX63430]: Genetex, Irvine, CA; DGAT2 [CAT#:IMG‐30279]: Imgenex, San Diego, CA; CD36 was generously provided by Dr. Tandon (33)) overnight at 4°C. After TBST washes, membranes were incubated at room temperature with appropriate HRP‐conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) diluted in TBST/1% nonfat dry milk for 1h. Enhanced chemiluminescence (ThermoScientific) was used to detect signals, which were then quantified by densiometry (Fluorchem HD2, ProteinSimple, Santa Clara, CA).

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Western blotting Smad4 using Anti-Smad4/DPC4 Antibody from Merck Millipore.

Manufacturer protocol

Download the product protocol from Merck Millipore for Anti-Smad4/DPC4 Antibody below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Western blotting Smad4 using Anti-Smad4/DPC4 Antibody from Merck Millipore. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.