Western blot analysis of extracts from 3T3 cells, untreated or treated with 20% serum, using Phospho-S6 Ribosomal Protein (Ser235/236) Antibody #2211 (left), or the same antibody incubated with specific blocking peptide (right).Learn more about how we get our images
Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Phospho-S6 Ribosomal Protein (Ser235/236) (91B2) Rabbit mAb #4857 in the presence of control peptide (left) or Phospho-S6 Ribosomal Protein (Ser235/236) Blocking Peptide (right).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: 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
NOTE: Do not allow slides to dry at any time during this procedure.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
posted June 2005
revised March 2016
Protocol Id: 303
This peptide is used to block Phospho-S6 Ribosomal Protein (Ser235/236) (91B2) Rabbit mAb #4857, Phospho-S6 Ribosomal Protein (Ser235/236) Antibody #2211, Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) (XP) Rabbit mAb #4858, and Phospho-S6 Ribosomal Protein (Ser235/236) (M20B19) Mouse mAb #5006 reactivity.
The quality of the peptide was evaluated by reversed-phase HPLC and by mass spectrometry. The peptide blocks Phospho-S6 Ribosomal Protein (Ser235/236) (91B2) Rabbit mAb #4857, Phospho-S6 Ribosomal Protein (Ser235/236) Antibody #2211, Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) (XP) Rabbit mAb #4858, and Phospho-S6 Ribosomal Protein (Ser235/236) (M20B19) Mouse mAb #5006 signal in immunohistochemistry and western Blotting.
Use as a blocking reagent to evaluate the specificity of antibody reactivity in immunohistochemistry protocols. For immunohistochemistry, add twice the volume of peptide as volume of antibody used in 100 µl total volume. Incubate for a minimum of 30 minutes prior to adding the entire volume to the slide. Recommended antibody dilutions can be found on the product data sheet.
For western immunoblotting, add 10 µl of antibody and 10 µl of blocking peptide to 10 ml of antibody dilution buffer, and incubate at room
temperature for 30 minutes before allowing to react with the blot.
Supplied in 20 mM potassium phosphate (pH 7.0), 50 mM NaCl, 0.1 mM EDTA, 1 mg/ml BSA, 5% glycerol and 1% DMSO. Store at –20°C.
One way that growth factors and mitogens effectively promote sustained cell growth and proliferation is by upregulating mRNA translation (1,2). Growth factors and mitogens induce the activation of p70 S6 kinase and the subsequent phosphorylation of the S6 ribosomal protein. Phosphorylation of S6 ribosomal protein correlates with an increase in translation of mRNA transcripts that contain an oligopyrimidine tract in their 5' untranslated regions (2). These particular mRNA transcripts (5'TOP) encode proteins involved in cell cycle progression, as well as ribosomal proteins and elongation factors necessary for translation (2,3). Important S6 ribosomal protein phosphorylation sites include several residues (Ser235, Ser236, Ser240, and Ser244) located within a small, carboxy-terminal region of the S6 protein (4,5).
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