公爵IP 实验步骤(英文)需具备一定英文能力

2021年1月16日发(作者：彭耜)
Co-immunoprecipitaton of polyribosomes that possess a FLAG-tagged
ribosomal protein with a-FLAG agarose beads
Maria Eugenia Zanetti/Bailey-Serres Lab
Center for Plant Cell Biology, UC Riverside
This protocol is a modification of the version provided from SIGMA in the technical
bulletin of FLAG-tagged protein immunoprecipitation kit (Product number: FLAGIPT-
1). Extraction buffer and binding buffer were optimized to preserve polyribosomal
integrity without affecting antigen-antibody binding reaction. This protocol can be used
for isolation of polysomes of leaves of mature Arabidopsis thaliana that are transgenic
for tagged ribosomal proteins.
Polysomes extraction
1- Homogenize the tissue (previously pulverized to a fine powder with a mortar and
pestle under liquid nitrogen) with 2 volumes of Polysome Extraction Buffer
(PEB). For preparative immunoprecipitation use 2.5 ml of packed tissue and 5 ml
of PEB. Incubate mixture on ice for 10 min.
2- Transfer to Corex centrifugation tubes and Centrifuge at 16,000 x g for 10 min at
4
0
C.
3- Transfer the supernatant to a new Corex tube and centrifuge at 16,000 x g for 10
min at 4
0
C.
4- Transfer the supernatant to a new tube. This fraction is SN 16,000 x g. Determine
A
260
.
Preparation of the a-FLAG M2 agarose beads (Sigma)
1- Thoroughly suspend the a-FLAG M2 agarose gel (Sigma, product number A
2220) in the vial to make a uniform suspension of the resin. The ratio of
suspension to packed volume should be 2:er 200 ml of suspension (100 ml
of packed gel) to a 15ml-Falcon tube.
2- Centrifuge at 8,200 x g for 30 sec.
3- Remove the supernatant with a pipette and add 2 ml of wash buffer.
4- Centrifuge at 8,200 x g for 30 sec.
5- Remove the supernatant with pipette and wash one more time with 2 ml of wash
buffer before to continue with the co-immunoprecipitation.
Co- immunoprecipitation
1- Mix 250 to 300 units of A
260
of SN 16,000 x g with 100 ml (packed volume) of a-
FLAG M2 agarose beads in a 15 ml- Falcon tube. Bring volume to 5 ml with
binding buffer.
2- Incubate for 2 h at 4
0
C with gently shaking (Binding Step)
3- Centrifuge for 30 sec at 8,200 x g at 4
0
C.
1
4- Transfer the supernatant to a new tube. This is the Supernatant of the Co-IP or
unbound fraction.
5- Add 4 ml of binding buffer to the beads, mix by inverting the tube and centrifuge
for 30 sec at 8,200 x g at 4
0
C (First wash).
6- Remove the supernatant with pipette and add 4 ml of binding buffer. Incubate at 4
0
C for 5 min. with gently shaking (Second wash).
7- Centrifuge for 30 sec at 8,200 x g at 4
0
C.
8- Remove the supernatant with pipette and add 4 ml of wash buffer. Incubate at 4
0
C for 5 min. with shaking (Third wash)
9- Centrifuge for 30 sec at 8,200 x g at 4
0
C.
10- Remove the supernatant and add to the beads 300 to 400 ml of wash buffer
containing 200 ng/ml of 3X-FLAG peptide (Sigma, product number F 4799).
Incubate for 30 min at 4
0
C with shaking (Elution Step).
11- Centrifuge for 30 se at 8,200 x g at 4
0
C. Transfer the supernatant to a new tube.
This is the elution of the Co-IP.
RNA Extraction
Note: this RNA extraction protocol uses Qiagen RNeasy kit (Cat Num.74904)
1- Add 1 vol of 8 M guanidine-HCl to the Elution of the Co-IP and vortex for 3 min.
2- Add 1.5 vol of 100% Ethanol and vortex for 1 min.
3- Precipitate the RNA at –20
0
C overnight.
4- Centrifuge at 16,000 x g for 45 min
5- Remove supernatant and let the pellet dry for 20 min.
6- Prepare extraction buffer adding 10 ml of b-mercaptoethanol to 1 ml of RLT buffer
(from Qiagen RNeasy kit).
7- Resuspend the pellet in 450 ml of extraction buffer and vortex for 1 min.
8- Add 250 ml of 100% ethanol and mix by inverting the tube. Do not Vortex
9-Apply the sample into an RNeasy mini spin column (pink). Incubate for 3 min.
10-Centrifuge for 15 sec at 16,000 x g
11- Add 700 ml of RW1 buffer and centrifuge for 15 sec at 16,000 x g. Discharge the
flow through.
12- Add 500 ml of RPE buffer (Four volumes of ethanol are already added) and
centrifuge for 15 sec at 16,000 x g. Discharge the flow through.
13- Add 500 ml of RPE buffer to the column and centrifuge for 2 min at16,000 x g.
14- Transfer the column to a new 2 ml microfuge tube, and centrifuge for 1 min at 16,000
x g.
15-Transfer the column to a new 1.7 ml microfuge tube and add 50 ml of RNAse free
water. Incubate for 5 min.
16-Elute RNA centrifuging for 1 min. at 16,000 x g.
2