For Research Use Only.
Not for use in diagnostic procedures.

PM044
Lot 012~Page 1POLYCLONAL ANTIBODY

Anti-PIWIL2 (MILI) (Mouse) pAb
Code No.
PM044

Quantity
100 µLBACKGROUND: The Piwi/Argonaute protein family

plays important roles in stem-cell self-renewal, RNAsilencing and translational regulation in various organisms.
The family members are defined by conserved both PAZ(Piwi Argonaut and Zwille) and Piwi domains participate in
RNA binding. Piwi-interacting RNA (piRNA) is a class ofsmall RNA molecules that is expressed uniquely inmammalian testes and forms RNA-protein complexes withPiwi proteins. These small regulatory RNAs have been
implicatedinspermatogenesis,repressionofretrotransposon transposition in germline cells, epigenetic
regulation and positive regulation of translation and mRNAstability. MIWI (PIWIL1) and MILI (PIWIL2) are mouse
homologues of Piwi. MILI is expressed from primordial
germ cells (PGC) in embryo, whereas MIWI is detected
later from the mid-pachytene stage to early roundspermatids. Their expression overlaps in the mid-pachytene
and early round spermatid stage. The expression patterns
and knockout phenotypes of mammalian Piwi members
indicated MILI is an essential factor for meiotic
differentiation during spermatogenesis. It was also reported
that MILI (PIWIL2) is widely expressed in various tumors
and acts as an oncogene by inhibiting apoptosis andpromoting proliferation through the activation ofStat3/Bcl-xL pathway.

SOURCE: This antibody was purified from rabbit serum

using affinity column. The rabbit was immunized with thesynthetic peptide, VRKDREEPRSSLPDPS, corresponding
to mouse PIWIL2 (107-122 aa).

FormAffinity Purified

APPLICATIONS:

Western blotting; 1:1,000 for chemiluminescence detection
system.
Immunoprecipitation; 1 µL/sample
Immunohistochemistry; Not recommended**PM043 is suitable for this application.
Immunocytochemistry; Not tested
Flow cytometry; Not tested
RNP immunoprecipitation (RIP); Reference 2)-4)
Detailed

procedure

PROTOCOLS.

isprovided

inthefollowing

INTENDED USE:

For Research Use Only. Not for use in diagnostic procedures.

REFERENCES:

1) Shiromoto, Y., et al., RNA 19, 803-810 (2013)
2) Kuramochi-Miyagawa, S., et al., Genes Dev. 24, 887-892 (2010) [RIP]3) Kojima, K., et al., Genes Cells. 14, 1155-1165 (2009) [WB, IP, RIP]
4) Kuramochi-Miyagawa, S., et al., Genes Dev. 22, 908-917 (2008) [RIP]
5) Aravin, A. A., et al., Science 316, 744-747 (2007)
6) Aravin, A., et al., Nature 442, 203-207 (2006)
7) Kuramochi-Miyagawa, S., et al., Development 131, 839-849 (2004)
8) Kuramochi-Miyagawa, S., et al., Mech. Dev. 108, 121-133 (2001)
This antibody is used in reference number 1)-4).

FORMULATION: 100 µL volume of PBS containing
50% glycerol, pH 7.2. No preservative is contained.

STORAGE: This antibody solution is stable for one year
from the date of purchase when stored at -20°C.

REACTIVITY: This antibody reacts with MILI on
Western blotting and Immunoprecipitation.

SPECIES CROSS REACTIVITY:
Species

Human

Mouse

Rat

TissueNot Tested

testisNot Tested

Reactivity on WB+MEDICAL & BIOLOGICAL LABORATORIES CO., LTD.

URL https://ruo.mbl.co.jp e-mail support@mbl.co.jp

PM044
Lot 012~Page 2The descriptions of the following protocols are examples.
Each user should determine the appropriate condition.

PROTOCOLS:SDS-PAGE & Western Blotting1) Wash the cells 3 times with PBS and suspend with 10
volume of cold Lysis buffer (50 mM Tris-HCl, pH 7.2,
150 mM NaCl, 0.1% NP-40, 2 mM EDTA, 10% glycerol)containing appropriate protease inhibitors. Incubate it at4oC with rotating for 30 minutes, then sonicate briefly (upto 10 seconds).
2) Centrifuge the tube at 12,000 x g for 10 minutes at 4°C
and transfer the supernatant to another tube. Measure theprotein concentration of the supernatant and add the coldLysis buffer to make 8 mg/mL solution.
3) Mix the sample with equal volume of Laemmli’s samplebuffer.
4) Boil the samples for 3 minutes and centrifuge. Load 10µL of the sample per lane in a 1-mm-thick
SDS-polyacrylamide gel for electrophoresis.
5) Blot the protein to a polyvinylidene difluoride (PVDF)
membrane at 1 mA/cm2 for 1 hour in a semi-dry transfer
system (Transfer Buffer: 25 mM Tris, 190 mM glycine,
20% MeOH). See the manufacture's manual for precisetransfer procedure.
6) To reduce nonspecific binding, soak the membrane in 5%skimmed milk/TBS-T (0.05% Tween-20 in TBS, pH 7.2)
for 1 hour at room temperature, or overnight at 4oC.
7) Incubate the membrane with primary antibody diluted
with TBS-T, pH 7.2 containing 1% skimmed milk assuggest in the APPLICATIONS for 1 hour at roomtemperature. (The concentration of antibody will dependon condition.)
8) Wash the membrane with TBS-T (5 minutes x 3 times).
9) Incubate the membrane with the 1:10,000 anti-IgG(Rabbit) pAb-HRP (MBL; code no. 458) diluted with 1%skimmed milk (in TBS, pH 7.2) for 1 hour at room
temperature.
10) Wash the membrane with TBS-T (10 minutes x 3 times).
11) Wipe excess buffer on the membrane, then incubate itwith appropriate chemiluminescence reagent for 1 minute.
12) Remove extra reagent from the membrane by dabbing
with paper towel, and seal it in plastic wrap.
13) Expose to an X-ray film in a dark room for 3 minutes.
14) Develop the film as usual. The condition for exposure anddevelopment may vary.

Immunoprecipitation

1) Wash the sample 2 times with PBS and homogenate withcold Lysis buffer (50 mM HEPES-KOH, pH 7.5, 150 mM
NaCl, 0.1% NP-40, 5 mM EDTA, 10% glycerol)containing appropriate protease inhibitors.
2) Centrifuge the tube at 12,000 x g for 10 minutes at 4°C
and transfer the supernatant to another tube.
3) Add 50 µL of 50% protein A agarose beads in thesupernatant. Incubate it at 4°C with rotating for 30-60minutes.
4) Centrifuge the tube at 12,000 x g for 5 minutes at 4°C.
Supernatant is equally divided into another two tube.
5) Add the normal rabbit IgG or anti-MILI antibody at theamount of as suggest in the APPLICATIONS to thesupernatant. Vortex briefly and incubate with gently
agitation for 90-120 minutes at 4°C.
6) Add 20 µL of 50% protein A agarose beads into the tube.
Mix well and incubate with gentle agitation for 30-60
minutes at 4°C.
7) Wash the beads 3-5 times with ice-cold Lysis buffer(centrifuge the tube at 2,500 x g for 10 seconds).
8) Resuspend the beads in 30 µL of Laemmli’s sample buffer,boil for 3-5 minutes, and centrifuge for 5 minutes. Use 15µL/lane for the SDS-PAGE analysis.
(See SDS-PAGE & Western blotting.)(Positive control for Immunoprecipitation; Mouse testis)

RELATED PRODUCTS:

Please visit our web site https://ruo.mbl.co.jp/(Positive control for Western blotting; Mouse testis)211116-4