Anti-PURA antibody

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-PURA antibody (AB79936)

ICC/IF image of ab79936 stained HeLa cells. The cells were 100% Methanol fixed (5 min) and then incubated in 1%BSA / 10% normal Goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab79936, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 Goat anti-Rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 100% Methanol fixed (5 min) Hek293. HepG2, MCF-7 cells at 5µg/ml.

• WB

Unknown

Western blot - Anti-PURA antibody (AB79936)

PURA contains a number of potential phosphorylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted. Furthermore, other commercially available antibodies react with a target at approximately 42 kDa.

All lanes:

Western blot - Anti-PURA antibody (ab79936) at 1 µg/mL

All lanes:

Human brain tissue lysate - total protein (<a href='/en-us/products/unavailable/human-brain-tissue-lysate-total-protein-ab29466'>ab29466</a>) at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 35 kDa

Observed band size: 42 kDa

true

• WB

CiteAb

Western blot - Anti-PURA antibody (AB79936)

PURA western blot using anti-PURA antibody ab79936. Publication image and figure legend from Moldovan, J. B. & Moran, J. V., 2015, PLoS Genet, PubMed 25951186.

ab79936 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab79936 please see the product overview.

The identification of host proteins immunoprecipitated with L1 ORF1p-FLAG.(A) Schematic of L1 constructs : pJM101/L1.3 expresses a human L1 (L1.3) [5] containing an mneoI retrotransposition indicator cassette within the L1 3' UTR. The pJM101/L1.3FLAG construct is identical to pJM101/L1.3, but contains a single FLAG epitope on the carboxyl-terminus of ORF1p. Both constructs were cloned into a pCEP4 mammalian expression vector. A CMV promoter augments L1 expression and an SV40 polyadenylation signal (pA) is located downstream of the native L1 polyadenylation signal. (B) Results of immunoprecipitation experiments : Whole cell lysates from HeLa cells transfected with either pJM101/L1.3 or pJM101/L1.3FLAG were subjected to immunoprecipitation using an anti-FLAG antibody. The proteins then were separated by SDS-PAGE, visualized by silver staining, and subjected to LC-MS/MS. An ~40 kDa band corresponding to the theoretical molecular weight of ORF1p is visible in the pJM101/L1.3FLAG lane (*). Black bars indicate the approximate molecular weights of the ORF1p-FLAG interacting proteins. Molecular weight standards (kDa) are shown on the left hand side of the gel. (C) Validation of the ORF1p-FLAG immunoprecipitation : Western blot experiments using an antibody specific to amino acids 31–49 of L1.3 ORF1p verified the enrichment of ORF1p-FLAG in pJM101/L1.3FLAG, but not pJM101/L1.3 immunoprecipitation reactions. Cells transfected with the pCEP4 vector served as a negative control. (D) Validation of putative ORF1p-FLAG interacting proteins : Western blot images of the pJM101/L1.3FLAG and pJM101/L1.3 immunoprecipitation (IP) reactions. The pCEP4 lanes denote whole cell lysates derived from HeLa cells transfected with an empty pCEP4 vector (~ 1.0% input). Primary antibodies used to probe western blots are indicated to the left of the images. Immunoprecipitation reactions were conducted in either the absence (left) or presence (right) of RNaseA (10 μg/mL). The putative cellular functions of the ORF1p-FLAG interacting proteins are indicated on the right hand side of the blots.

false