Anti-RPL5 antibody

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RPL5 antibody (AB86863)

IHC image of RPL5 staining in human liver carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab86863, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-RPL5 antibody (AB86863)

ICC/IF image of ab86863 stained HepG2 cells. The cells were 4% PFA fixed (10 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 (ab86863, 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 4% PFA fixed (10 min) HeLa cells at 5µg/ml, and in 100% methanol fixed (5 min) HeLa, HepG2 and MCF7 cells at 5µg/ml

• IP

Unknown

Immunoprecipitation - Anti-RPL5 antibody (AB86863)

RPL5 was immunoprecipitated using 0.5mg HepG2 whole cell extract, 5µg of Rabbit polyclonal to RPL5 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, HepG2 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab86863.
Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band : 34kDa, non specific band - 68kDa as present in test (lane 1) and control (lane 2); 68kDa : We are unsure as to the identity of this extra band; RPL5

All lanes:

Immunoprecipitation - Anti-RPL5 antibody (ab86863)

Predicted band size: 34 kDa

false

• WB

Unknown

Western blot - Anti-RPL5 antibody (AB86863)

All lanes:

Western blot - Anti-RPL5 antibody (ab86863) at 1 µg/mL

Lane 1:

Liver (Human)Tissue Lysate - Fetal tissue at 10 µg

Lane 2:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 3:

MEL-1 (Human embryonic stem cell, male cell line) Whole Cell Lysate (<a href='/en-us/products/unavailable/mel-1-human-embryonic-stem-cell-male-cell-line-whole-cell-lysate-ab27198'>ab27198</a>) at 10 µg

Lane 4:

Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 34 kDa

Observed band size: 34 kDa,68 kDa

true

Exposure time: 1min

• WB

CiteAb

Western blot - Anti-RPL5 antibody (AB86863)

RPL5 western blot using anti-RPL5 antibody ab86863. Publication image and figure legend from Hao, Q., Wang, J., et al., 2020, Cell Death Dis, PubMed 32198344.

ab86863 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 ab86863 please see the product overview.

Ablation of endogenous SBDS induces RPL5- and RPL11-dependent p53 stabilization.a, b Knockdown of SBDS prolongs p53 protein half-life. HCT116p53+/+ cells were transfected with control or SBDS siRNA for 48–72 hr. CHX was supplemented into the medium for the indicated time before the cells were harvested for IB analysis a. Quantification of the p53/β-actin ratios is shown in the b. c RPL5 and RPL11 are required for SBDS depletion-induced p53 activation. HCT116p53+/+ cells were transfected with the indicated siRNAs for 48–72 hr and harvested for IB analysis using the antibodies as indicated. d Knockdown of SBDS enhances the RPL11-MDM2 interaction. HCT116p53+/+ cells were transfected with control or SBDS siRNA for ~48 hr. The proteasome inhibitor MG132 was supplemented into the medium for 4 hr before the cells were harvested for co-IP-IB assays using antibodies as indicated. e Knockdown of SBDS enhances the RPL5-MDM2 interaction. The same experiments were conducted as described in d, except that the anti-RPL5 antibody was used.

false