Protein detection by immunoblotting was performed using antibodies raised against Fancd2 (Abcam, ab108928, 1/500 dilution), Fanci (Abcam, ab74332, 1/500), Fancr (Calbiochem, PC130, 1/2,500), E2F4 (Santa Cruz, C-20, 1/200), p53 (Novocastra, CM5, 1/1,000), p21 (Santa Cruz, F-5, 1/250) or actin (Santa Cruz, C-4, 1/5,000) and chemiluminescence revelation was achieved with SuperSignal west dura (Perbio, France). Band quantification was performed using ImageJ, with actin as loading control. Control bands for Fancd2, Fanci and Fancr proteins were obtained using 20 μl lipofectamine 2000 according to the supplier's procedure to transfect NIH-3T3 cells (a 10-cm dish at 60% confluency) with 3 μg of a pCAGGS expression vector (empty pCAGGS vector, pCAGGS-Fancd2, pCAGGS-Fanci or pCAGGS-Fancr—cloning details upon request), then extracting proteins after 24 h. Uncropped scans of the western blots in Figs 1d and 4b , as well as relevant controls, are presented in the Supplementary Figs 2 and 9 , respectively.
Pc130
Manufactured by Santa Cruz Biotechnology
Sourced in France
The PC130 is a high-performance centrifuge designed for a variety of laboratory applications. It features a compact and durable construction, allowing for efficient sample processing. The centrifuge can accommodate a range of rotor options to accommodate different sample volumes and tube sizes.
Automatically generated - may contain errors
Lab products found in correlation
Ab108928, by Abcam (1 mentions)
Fluorescent mounting medium, by Agilent Technologies (1 mentions)
Axiovision, by Zeiss (1 mentions)
Axio imager z1 microscope, by Zeiss (1 mentions)
Peroxidase linked anti mouse or anti rabbit igg, by GE Healthcare (1 mentions)
T9026, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
Mouse anti γh2ax, by Merck Group (1 mentions)
Sc 8973, by Santa Cruz Biotechnology (1 mentions)
4 protocols using pc130
1
Western Blot Analysis of DNA Repair Proteins
2
Immunofluorescence assay for RAD51 and PCNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were pre-extracted in CSK100 buffer for 5 min on ice under gentle agitation. Cells were fixed in 4% paraformaldehyde for 20 min and permeabilized in methanol at –20°C for 10 s. Cells were incubated for 1 h at room temperature with primary antibodies (rabbit anti-RAD51 Calbiochem PC130 1/200 and mouse anti-PCNA Santa Cruz PC10 1/500) diluted in IF buffer (3% BSA, 0.5% Tween 20 in PBS). Cells were washed three times in PBS and stained for 30 min with secondary antibodies from Molecular Probes (goat anti-rabbit AF488 1/1000, goat anti-mouse AF594 1/1000). Coverslips were mounted in fluorescent mounting medium (DAKO) supplemented with DAPI (1.5 μg/ml). Images were acquired on an AxioImager Z1 microscope using the Axio Vision software (Zeiss). Intensity was quantified with the ImageJ software.
3
Extracting Testis Proteins for Immunoblotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To prepare testis extracts, adult mouse testes were macerated in NETN buffer (100 mM NaCl, 20 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 0.5% (v/v) Nonidet P-40 (NP-40)) with protease inhibitor cocktail (Roche) and incubated for 30 min at 4 °C with gentle flicking every few minutes to resuspend the extracts in the buffer. The supernatant was collected after spinning the samples at 20,000×g for 10 min at 4°C. Thirty micrograms of protein were loaded on a precast SDS PAGE gel (Bio-Rad), transferred onto nitrocellulose membrane (162-0145, Bio-Rad), and blocked with 5% milk in PBST (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween 20) for 1 h. For immunodetection, the following antibodies were used: rabbit anti-RAD51 (Calbiochem, PC130; 1:5000), mouse anti-DMC1 (Santa Cruz Biotechnology, sc-373862; 1:500) and mouse anti-tubulin (Sigma, T9026; 1:10,000). Secondary antibodies used were peroxidase-linked anti-mouse or anti-rabbit IgG (1:10,000; GE Healthcare).
4
Immunofluorescence Analysis of Spermatocyte Spreads
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence experiments on surface spread spermatocytes were carried out as previously described28 (link). In brief, cells were permeabilized for 10 min in 0.05% Triton X-100 and fixed for 1 h minimum in 2% formaldehyde, 0.02% SDS in phosphate-buffered saline (PBS). Slides were rinsed in distilled water, air dried and blocked in PBT (0.15% bovine serum albumin, 0.10% TWEEN-20 in PBS) for 1 h. Slides were incubated with the following antibodies in a humid chamber overnight at 37 °C: guinea-pig anti-SYCP3 (made in-house) 1:500, rabbit anti-SYCP3 (Abcam ab-15092) 1:100, rabbit anti-ATR (Cell Signalling #2790) 1:50, mouse anti-γH2AX (Millipore 05–636) 1:100, rabbit anti-HORMAD2 (Tóth lab) 1:100, guinea-pig anti-SYCE2 (gift from Howard Cooke/Ian Adams) 1:800, human anti-centromere (CREST ab gift from Bill Earnshaw) 1:1000, rabbit anti-RPA (Abcam ab-2175) 1:100 anti-rabbit RAD51 (Calbiochem PC130) 1:25, goat anti-DMC1 (Santa Cruz sc-8973) 1:25. Western blotting was carried out as previously described77 (link) (Supplementary Fig. 6 ). Counting of recombination protein foci was performed manually, considering only foci that colocalized with axial elements.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!