REACTIVITY | SENSITIVITY | MW (kDa) | Isotype |
---|---|---|---|
H | Endogenous | 32 | Rabbit IgG |
Western blot analysis of extracts from HeLa and 293 cells, untreated or treated with UV (100 mJ/cm2, 2 hr recovery), using Phospho-RPA32 (Ser8) (E5A2F) Rabbit mAb (upper) or total RPA32/RPA2 Antibody #52448 (lower).
Learn more about how we get our images.Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with UV (100 mJ/cm2, 2 hr recovery; right), using Phospho-RPA32/RPA2 (Ser8) (E5A2F) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 594 Phalloidin #12877 (red). Blue = DAPI #4083 (fluorescent DNA dye).
Learn more about how we get our images.Flow cytometric analysis of 293T cells, untreated (blue) or treated with UV (100 mJ/cm2, 4 hr recovery; green), using Phospho-RPA32 (Ser 8) (E5A2F) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Learn more about how we get our images.For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Loading of prestained molecular weight markers (#13953, 5 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our #12727 Immunofluorescence Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted November 2006
revised November 2013
Protocol Id: 24
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised June 2017
Protocol Id: 404
Application | Dilutions |
---|---|
Western Blotting | 1:1000 |
Immunofluorescence (Immunocytochemistry) | 1:200 - 1:800 |
Flow Cytometry | 1:800 |
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
Phospho-RPA32/RPA2 (Ser8) (E5A2F) Rabbit mAb recognizes endogenous levels of RPA32/RPA2 protein only when phosphorylated at Ser8.
Human
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser8 of human RPA32/RPA2 protein.
RPA70 (HSSB, REPA1, RF-A, RP-A, p70) is a component of a heterotrimeric complex, composed of 70, 32/30 and 14 kDa subunits, collectively known as RPA. RPA is a single stranded DNA binding protein, whose DNA binding activity is believed to reside entirely in the 70 kDa subunit. The complex is required for almost all aspects of cellular DNA metabolism such as DNA replication (1-3), recombination, cell cycle and DNA damage checkpoints, and all major types of DNA repair including nucleotide excision, base excision, mismatch and double-strand break repairs (4-7). In response to genotoxic stress in eukaryotic cells, RPA has been shown to associate with the Rad9/Rad1/Hus1 (9-1-1) checkpoint complex (8). RPA is hyperphosphorylated upon DNA damage or replication stress by checkpoint kinases including ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK) (9-11). Phosphorylation of RPA32 occurs at serines 4, 8 and 33 (11). Hyperphosphorylation may alter RPA-DNA and RPA-protein interactions. In addition to the checkpoint partners, RPA interacts with a wide variety of protein partners, including proteins required for normal replication such as RCF, PCNA and Pol α, and also proteins involved in SV40 replication, such as DNA polymerase I and SV40 large T antigen (10,12).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc. Alexa Fluor is a registered trademark of Life Technologies Corporation. DyLight is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries. Tween is a registered trademark of ICI Americas, Inc.
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Product # | Size | Price |
---|---|---|
54762S | 100 µl | N/A |
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