Anti-PCNA antibody - Proliferation Marker (ab18197)

Western blot

All lanes : Anti-PCNA antibody - Proliferation Marker (ab18197) at 1 µg/ml

Lane 1 : HEK293 lysate
Lane 2 : HEK293 lysate with PCNA peptide (ab18602) at 1 µg/ml

Lysates/proteins at 20 µg/ml per lane.

Secondary
Alexa Fluor Goat-anti-Rabbit IgG (700) at 1/10000 dilution

Predicted band size : 29 kDa
Observed band size : 29 kDa
Additional bands at : 48 kDa (possible cross reactivity),75 kDa (possible cross reactivity).

Immunocytochemistry/ Immunofluorescence - PCNA antibody (ab18197)

ICC/IF image of ab18197 stained NIH/3T3 cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab18197, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

Immunocytochemistry/ Immunofluorescence - PCNA antibody (ab18197)

ab18197, at a 1/5000 dilution, staining PCNA in assynchronous HeLa cells. Cells were counter-stained with DAPI (red). For more information please refer to Abreview.

This image is courtesy of an Abreview submitted by Dr Kirk McManus

Western blot - PCNA antibody (ab18197)

All lanes : Anti-PCNA antibody - Proliferation Marker (ab18197) at 1 µg/ml

Lane 1 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate (ab7179)
Lane 2 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size : 29 kDa
Observed band size : 29 kDa
Additional bands at : 48 kDa (possible cross reactivity),52 kDa. We are unsure as to the identity of these extra bands.

Immunocytochemistry/ Immunofluorescence - PCNA antibody (ab18197)

ab18197, at a 1/2000 dilution staining PCNA in MRC5 Sv40 transformed fibroblasts. Cells were counterstained with DAPI (blue). For more information please refer to Abreview.

Dr Catherine Green

Immunoprecipitation - PCNA antibody (ab18197)

ab18197 staining rat PC12 whole cell lysate by Immunoprecipitation. 

ab18197 was incubated with the lysate (at a concentration of 5µg/ml) and a Protein A matrix for 12 hours at 4°C to achieve immunoprecipitation.  400µg of protein were present in the lysate input.

Lane order: PCNA IP (lane 1), Control IP (lane 2), Input 5% (lane 3)

This antibody immunoprecipitates a product of ~34 kDa which is consistent with the mobility of PCNA on SDS-PAGE

ab18197 was also used for the western blot step, at a contration of 1µg/ml

This image is courtesy of an Abreview submitted by Antibody Solutions Ltd

Immunocytochemistry/ Immunofluorescence - PCNA antibody - Proliferation Marker (ab18197)

ICC/IF image of ab18197 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 (ab18197, 1µg/ml) overnight at +4ºC. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) used at a 1/250 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.

Western blot

Anti-PCNA antibody - Proliferation Marker (ab18197) at 1 µg/ml + PCNA protein (His tag) (ab85651) at 0.01 µg

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP), pre-adsorbed (ab97080) at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Exposure time : 10 seconds

Immunocytochemistry/ Immunofluorescence - Anti-PCNA antibody - Proliferation Marker (ab18197)

ab18197 staining PCNA in Zebrafish gastrula embryos by Immunocytochemistry/ Immunofluorescence (wholemount).

Zebrafish embryos were fixed overnight at 4°C when they had reached 60% epiboly. Cells were fixed in formaldehyde, permeabilized using Proteinase K, blocked with 2% goat serum for 2 hours at 20°C and then incubated with ab18197 at a 1/500 dilution for 16 hours at 4°C. The secondary used was an Alexa-Fluor 488 conjugated goat anti-rabbit polyclonal used at a 1/500 dilution. Cells were post-fixed in PFA for 20 minutes at room temperature after extensive washing of the secondary antibody.

The left panel shows DAPI stained nuclei, the center panel is PCNA staining, and the right panel is the merged image.

Image courtesy of Hank Farr by Abreview.