Anti-MCM3 抗体 (ab4460)

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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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.