Ab4459

Manufactured by Abcam

Sourced in Spain, United States

Ab4459 is an anti-Human IgG antibody that can be used for various immunological applications. It is a purified recombinant antibody produced in HEK293 cells.

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4 protocols using ab4459

Antibody Characterization for Cell Biology

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The following antibodies were used for Western blotting:

RIF1; Bethyl Laboratories; A300‐568A; rabbit polyclonal

GFP; Abcam; ab290; rabbit polyclonal

GFP; ChromoTek; rat monoclonal [3H9]

MCM4; Abcam, ab4459; rabbit polyclonal

p‐S40‐MCM2; Abcam; ab133243; rabbit monoclonal [EPR4170(2)]

p‐S53‐MCM2; Bethyl Laboratories; A300‐756A; rabbit polyclonal

PP1α; Santa Cruz Biotechnology; sc‐271762; mouse monoclonal [G‐4]

PP1β; Santa Cruz Biotechnology; sc‐373782; mouse monoclonal [C‐5]

PP1γ; Santa Cruz Biotechnology; sc‐6108; goat polyclonal

Tubulin; Santa Cruz Biotechnology; sc‐53030; rat monoclonal [YOL1/34]

Following antibodies were used for flow cytometric analysis of chromatin‐associated proteins at indicated dilutions:

Cdt1 antibody; Abcam ab202067, at 1/100 dilution

FLAG tag antibody [M2]; Sigma F‐1804, at 1/300 dilution

MCM3 antibody; Santa Cruz sc‐9850, at 1/300 dilution

ORC2 antibody; Abcam ab31930, at 1/200 dilution

Alexa Fluor 488‐conjugated anti‐mouse IgG (ab150109), at 1/2,000 dilution

Alexa Fluor 568‐conjugated anti‐mouse IgG (ab175700), at 1/2,000 dilution

Alexa Fluor 647‐conjugated anti‐goat IgG (ab150135), at 1/2,000 dilution

Alexa Fluor 647‐conjugated anti‐rabbit IgG (ab150063), at 1/2,000 dilution

For immunoprecipitation of endogenous RIF1 protein, Abcam ab70254 was used. For microscopic observation of Cdc6 protein, Abcam ab188423 was used.

Hiraga S., Ly T., Garzón J., Hořejší Z., Ohkubo Y., Endo A., Obuse C., Boulton S.J., Lamond A.I, & Donaldson A.D. (2017). Human RIF1 and protein phosphatase 1 stimulate DNA replication origin licensing but suppress origin activation. EMBO Reports, 18(3), 403-419.

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Characterization of MCM Protein Expression

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For WB, the detailed procedures were performed as described previously [8] using the following primary antibodies: anti-MCM2 (Abcam, ab4461), anti-MCM3 (Abcam, ab128923), anti-MCM4 (Abcam, ab4459), anti-MCM5 (Abcam, ab75975), anti-MCM6 (Abcam, ab201683), anti-MCM7 (Abcam, ab2360), anti-CDK9 (Abcam, ab76320), anti-CyclinD1 (Abcam, ab16663), anti-CyclinE1 (Abcam, ab33911), anti-p53 (Abcam, ab241566), anti-p21 (Abcam, ab109502), anti-p27 (Abcam, ab32034), and anti-GAPDH (Abcam, ab181602).
For IHC, the standard method was described previously.14 (link) Slides were incubated with primary antibodies (Abs) against MCM2 (Immuway, YM6642), MCM3 (Abcam, ab128923), MCM4 (Immuway, YT2681), MCM5 (Abcam, ab75975), MCM6 (Abcam, ab190948), and MCM7 (Abcam, ab2360).

Niu Y., Shan L., Gao H., Zhang C., Qian Z., Wang Z., Xu X., Zhang X., Wang J., Ma L., Chen L, & Yu Y. (2020). Huaier Suppresses the Hepatocellular Carcinoma Cell Cycle by Regulating Minichromosome Maintenance Proteins. OncoTargets and therapy, 13, 12015-12025.

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Western Blot Analysis of Cell Signaling Proteins

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Proteins (20–50 µg) were loaded on a polyacrylamide gel and then transferred onto Immobilon-P. Membranes were incubated with homemade rabbit polyclonal antibodies raised against recombinant full length 6His-human Ensa protein (anti-hEnsa) (1/1000), phospho-Arpp19/Ensa S67/S71 (Cell Signaling, #5240, 1/250,), human Greatwall (anti-hGreatwall) (1/1000), phospho-Greatwall S875^{34 (link)}, α-Tubulin (1/1000 clone C102, generous gift of J.M. Andreu, Spain), Treslin (1/1000, Bethyl Laboratories Inc., A303-472A), phospho-Ser CDK substrate (1/1000, Cell Signalling, #9477S), Histone H3 (1/500, Santa Cruz, Biotechnology Inc., SC-FL136), Cdk1 (1/1000, Santa Cruz, Biotechnology Inc., SC-954), MCM-2 (1/10000, Abcam, ab4461), MCM-3 (1/2500, Abcam, ab4460), MCM-4 (1/5000, Abcam, ab4459) and MCM-5 (1/5000, Abcam, ab17967). Mouse monoclonal used are MCM-7 antibody (1/1000, Abcam, ab2360), Flag M2 (1/500, Sigma, F3165), Vinculin (1/1000, Sigma,V9131 Hvin-1) and β-Tubulin (1/500, clone E7, generous gift from N. Morin). Uncropped scans of all the western blot membranes are supplied in the Supplementary Information file.

Charrasse S., Gharbi-Ayachi A., Burgess A., Vera J., Hached K., Raynaud P., Schwob E., Lorca T, & Castro A. (2017). Ensa controls S-phase length by modulating Treslin levels. Nature Communications, 8, 206.

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Quantifying MCM Proteins by Western Blot

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The primary antibodies used in this study against MCM2 (ab31159), MCM3 (ab128923), MCM4 (ab4459), MCM5 (ab75975), MCM6 (ab201683), MCM7 (ab52489), and GAPDH (ab181603) were obtained from Abcam (Cambridge, MA, USA). Western blotting was conducted according to our previous report [56 (link)].

Cao T., Yi S.J., Wang L.X., Zhao J.X., Xiao J., Xie N., Zeng Z., Han Q., Tang H.O., Li Y.K., Zou J, & Wu Q. (2020). Identification of the DNA Replication Regulator MCM Complex Expression and Prognostic Significance in Hepatic Carcinoma. BioMed Research International, 2020, 3574261.

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