Total protein concentration for standardized plasma membrane preparations of Madin-Darby canine kidney (MDCK) and multidrug-resistant (MDR) MDCK cells was determined using a Pierce BCA Protein Assay Kit according to the manufacturer’s instructions. Total protein (30 μg) isolated from MDCK or MDR MDCK cells was used as a positive control. Similar preparations were made from B16, TC2, and 4T1 cells at low (2.5 × 105–5 × 105) and high (1.5 × 107–2 × 107) cell counts. The samples were separated on a 4%–15% Tris–HCl gradient polyacrylamide gel and electrotransferred to polyvinylidene fluoride membrane. The membrane was incubated in blocking buffer (5% nonfat dry milk in PBS with 0.1% Tween 20) for 1 h at room temperature. The membrane was then incubated with P-gp detection antibody C219 (Thermo Fisher) overnight at 4 °C, washed (0.1% Tween 20 in PBS), and incubated with alkaline phosphatase-conjugated secondary antibody (Promega) for 30 min at 25 °C. The membrane underwent a final wash before staining using Immune Star AP substrate (Bio-Rad) according to the manufacturer’s instructions (Fig. 4c ).
Immune star ap substrate
Manufactured by Bio-Rad
The Immune Star AP substrate is a chemiluminescent detection reagent for Western blotting assays. It is used to visualize alkaline phosphatase (AP)-conjugated secondary antibodies on immunoblots.
Automatically generated - may contain errors
Lab products found in correlation
Alkaline phosphatase conjugated secondary antibody, by Promega (1 mentions)
Ip lysis buffer, by Thermo Fisher Scientific (1 mentions)
Anti p300, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Mini protean tgx, by Bio-Rad (1 mentions)
Turbofectin 8, by OriGene (1 mentions)
2 protocols using immune star ap substrate
1
Quantification of P-glycoprotein in Cell Lines
2
Immunoprecipitation and Western Blotting Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Saos-2 cells were seeded (106 cells/well) in 6-cm plates 24 h prior to transfection. DNA (2 μg) was transfected in each plate with Turbofectin 8.0 (Origene). At 48 h, plates were chilled on ice, washed, and lysed with IP Lysis Buffer (Thermo Scientific, 87,787) supplemented with protease inhibitor cocktail (Sigma-Aldrich, S8820). After centrifugation, cell lysates were incubated with agarose conjugates with anti-HA (MBL, 561–8, rabbit IgG), anti-Myc (Abcam, Ab1253, goat IgG), or anti-p300 (Santa Cruz, sc-48,343, mouse IgG) for 2 h at 4 °C. Protein complexes were washed four times, and dissociated in SDS-PAGE sample buffer. We used 7.5% polyacrylamide gel (Mini-PROTEAN TGX, Bio-Rad) for SDS-PAGE. Anti-Myc (MBL, 562), anti-p63 (Abcam, Ab735), anti-p300 (Santa Cruz, sc-585), anti-NFYC (Santa Cruz, sc-390,861), anti-Flag (Sigma, F3165, M2), anti-E1A (Santa Cruz, sc-58,658) and anti-HDAC1 (Abcam, ab19845) antibodies were used for western blotting. Alkaline phosphatase-conjugated secondary antibodies (Cell Signaling Technology, #7056S and #7054S) were used in combination with Immune-Star AP Substrate (Bio-Rad, 1,705,018).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!