Overview

  • Product name
    Anti-MCM3 antibody - ChIP Grade
    See all MCM3 primary antibodies
  • Description
    Rabbit polyclonal to MCM3 - ChIP Grade
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ICC/IF, IP, WB, ChIP, IHC-Pmore details
  • Species reactivity
    Reacts with: Chicken, Human
    Predicted to work with: Mouse, Horse, Pig, Chimpanzee, Rhesus monkey, Gorilla, Orangutan
  • Immunogen

    Synthetic peptide (Human) - 38 amino acids which represent a portion of human MCM 3 encoded in part by exons 10 and 11.

Properties

Applications

Our Abpromise guarantee covers the use of ab4460 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ICC/IF Use a concentration of 1 µg/ml.
IP Use at 2-5 µg/mg of lysate.
WB 1/2000 - 1/10000. Detects a band of approximately 105 kDa (predicted molecular weight: 90 kDa).
ChIP Use at an assay dependent concentration. PubMed: 19073720
IHC-P 1/1000 - 1/5000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Target

  • Function
    Acts as component of the MCM2-7 complex (MCM complex) which is the putative replicative helicase essential for 'once per cell cycle' DNA replication initiation and elongation in eukaryotic cells. The active ATPase sites in the MCM2-7 ring are formed through the interaction surfaces of two neighboring subunits such that a critical structure of a conserved arginine finger motif is provided in trans relative to the ATP-binding site of the Walker A box of the adjacent subunit. The six ATPase active sites, however, are likely to contribute differentially to the complex helicase activity. Required for DNA replication and cell proliferation.
  • Sequence similarities
    Belongs to the MCM family.
    Contains 1 MCM domain.
  • Post-translational
    modifications
    Phosphorylated upon DNA damage, probably by ATM or ATR.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • Cervical cancer proto oncogene 5 antibody
    • DNA polymerase alpha holoenzyme associated P1 antibody
    • DNA polymerase alpha holoenzyme associated protein P1 antibody
    • DNA polymerase alpha holoenzyme-associated protein P1 antibody
    • DNA replication factor MCM3 antibody
    • DNA replication licensing factor mcm3 antibody
    • HCC 5 antibody
    • HCC5 antibody
    • hRlf beta subunit antibody
    • Human cervical cancer proto oncogene 5 antibody
    • MCM 3 antibody
    • mcm3 antibody
    • MCM3 minichromosome maintenance deficient 3 antibody
    • MCM3_HUMAN antibody
    • MGC1157 antibody
    • Minichromosome maintenance complex component 3 antibody
    • Minichromosome maintenance deficient 3 antibody
    • Minichromosome maintenance protein 3 antibody
    • P1 h antibody
    • P1 MCM3 antibody
    • P1 Protein antibody
    • P1-MCM3 antibody
    • P1.h antibody
    • p102 antibody
    • P102 protein antibody
    • Replication licensing factor beta subunit antibody
    • RLF beta subunit antibody
    • RLF subunit beta antibody
    • RLFB antibody
    see all

Images

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon carcinoma tissue labelling MCM3 with ab4460 at 1/1000 (1µg/ml). Detection: DAB.
  • HeLa cell RIPA extract (50 µg) incubated with ab4460 used at 0.2 µg/ml for 1 hour. HeLa cell RIPA extract (50 µg) incubated with ab4460 used at 0.2 µg/ml for 1 hour.
  • WB using ab4460. Western blot analysis of ab4460 on HeLa OHIO extract.
  • ICC/IF image of ab4460 stained HeLa cells. The cells were 4% formaldehyde 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 (ab4460, 1µg/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.

References

This product has been referenced in:
  • Charrasse S  et al. Ensa controls S-phase length by modulating Treslin levels. Nat Commun 8:206 (2017). Read more (PubMed: 28785014) »
  • Zhou Y  et al. Role of WDHD1 in Human Papillomavirus-Mediated Oncogenesis Identified by Transcriptional Profiling of E7-Expressing Cells. J Virol 90:6071-84 (2016). WB ; Human . Read more (PubMed: 27099318) »
See all 19 Publications for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Answer

Thank you for contacting us.
I'm sorry to hear that the antibody does not seem to work in IP.

The protocol used to test the antibody in IP is as follows:

Preparation of Cell Lysate used for Immunoprecipitation

Reagents needed:

PBS (Phosphate Buffered Saline, pH 7.2)


NaCl 8.0 g
KCl 0.2 g
NaH2PO4 0.23 g
Na2HPO4 0.12 g



Adjust the volume to 1 liter with distilled water.

Store at 4-25 C.

IP Lysis Buffer


NaCl 7.31 g
1 M Tris, pH 8 25 ml
0.5 M EDTA, pH 8 5 ml
10% NP-40 25 ml
Distilled Water 445 ml



Store at 4 C.

Protease Inhibitors
Reconstitute with 1 ml of dH2O. Use 50 mcl per 5 ml of lysis buffer to harvest cells.

Procedure:

Allow cells to grow to approximately 80% confluence on 100mm polystyrene tissue culture plates. Approximately 8*10^6 cells (one plate at 80% confluence) are needed per IP reaction.
Wash cells two times in cold PBS.
Lyse cells to release soluble cellular proteins using 500 mcl of cold lysis buffer containing 1X protease inhibitors per 100 mm plate. Scrape cells from the plates, transfer to 1.5 ml micro-centrifuge tubes, and place on ice for 30 minutes to ensure efficient lysis.
Centrifuge lysates at 10,000 x g; 5 minutes.
Remove and pool the supernatant. Determine protein concentration.




Immunoprecipitation Protocol

Reagents Needed:

Immunoprecipitation (IP) lysis buffer
Protease Inhibitors
Primary Antibodies
Normal IgG, negative control
Protein A Sepharose Beads
Cell Lysate
Sample Buffer

IP lysis Buffer
12.5 ml 1M NaCl (250mM)
2.5 ml 1M Tris (50mM)
500ul 0.5M EDTA (5mM)
2.5ml 10% NP-40
32 ml dH20

Protein A Beads
Resuspend 400 mg of Protein A beads in 10 ml of distilled H2O. Mix well to resuspend. Spin at 250 rpm for 5 minutes. Wash 3X in 10 ml IP Lysis buffer. Resuspend to 10 ml with IP lysis buffer for a 20% solution. Use 100 mcl per IP reaction.

4X Sample Buffer
Glycerol 4.0 g
Tris Base 0.68 g
Tris HCL 0.67 g
LDS 0.80 g
EDTA 6 mg
Brilliant Blue G250 2.5 mg
Phenol red 2.5 mg
Adjust volume to 10 ml with ultra pure water.

Store at 4 C.

1X Sample Buffer
4X sample buffer 150 mcl
1M DTT 60 mcl
Distilled water 390 mcl

Make fresh for each use.

Procedure:

Prepare cell lysate according to protocol. Place 500 mcl of the prepared cell lysate (1-3 mg/ml) into a 1.5 ml micro-centrifuge tube.
To this tube add 2 to 10 mcg of the primary antibody (If using neat sera or an IgG fraction such as Protein-A purified antibody, larger amounts are likely to be required. For best results, optimal amounts of antibody should be empirically defined.)
To a negative control reaction, add an equivalent amount of normal rabbit IgG.
Add 100 mcl of a 20% Protein A suspension to the mixture of antibody and cell lysate. Rotate the immunoprecipitation reactions (end-to-end) for 3 hours at room temperature or overnight at 4 C.
Centrifuge (200 x g; 5 minutes) to pellet the complex.
Remove the supernatant and add 500 mcl cold cell lysis buffer. Centrifuge (200 x g; 5 minutes).
Repeat wash step 6 twice more. After each centrifugation remove as much of the supernatant as possible.
After removing the supernatant from the third wash, add 40 mcl of freshly prepared 1X sample buffer to each tube and heat at 90 C for 5 minutes.
Continue with electrophoresis and immunoblotting as described under western blotting protocol. Load 8 to 16 mcl (20 to 40% of the IP reaction) to a polyacrylamide gel.



Note: For optimal results, complete reduction of the sample is required. We recommend the use of 0.1 M DTT in SDS-PAGE sample buffer and immediately heating samples, loading and running gels.

Some additional advice and suggestions:

The antibody has been tested without conjugation to beads, thus I would suggest trying it that way as well. If the conjugation does not work efficiently, then the IP will not work either.

It is also important to include the isotype control (ab37415, https://www.abcam.com/Rabbit-IgG-ChIP-Grade-ab37415.html) as negative control as this would give you a better idea of the non-specific background.
I'd suggest trying various antibody-to-lysate ratios (e.g. 1/100 to 1/1000) as the optimal ratio needs to be determined empirically and can vary from lysate type to lysate type and from antibody to antibody.
Furthermore, in case that this might be a problem, for avoiding the heavy and light chain bands in the WB, I'd recommend using protein A/G-HRP (ab7456, ab7460) instead of the secondary antibody in the WB.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (HeLa)
Loading amount
50 µg
Specification
HeLa
Gel Running Conditions
Reduced Denaturing (12%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Nov 08 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (HeLa)
Loading amount
25 µg
Specification
HeLa
Gel Running Conditions
Reduced Denaturing (12%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Dr. Mirjam Eckert

Verified customer

Submitted Oct 30 2009

Answer

Thank you for your enquiry. There is a very low probability that ab3724 would react with Drosophila MCM3 and very low to zero probability that ab3725 or ab4460 would react too. I hope this information will be useful. Should you require any other assistance, please do not hesitate to contact me.

Read More

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