Optiblot SDS-PAGE Gel Instructions for Use For the use in SDS-PAGE with precast gels This product is for research use only and is not intended for diagnostic use.
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Table of Contents 1. Introduction 3 2. Storage 3 3. Sample and Running Buffer Preparation 3 4. Sample Loading 5 5. Run Conditions 6 6. Gel Staining 7 7. Gel Drying 8 8. Gel Blotting 9 9. Buffer Recipes 11 2
1. Introduction Optiblot precast gels offer superior rigidity and stability over traditional polyacrlyamide gels and are compatible with the most common tanks and systems. For convenience the precast gels come with the comb already removed as the cassette locks the fingers in place, for additional convenience there is no tape to be removed and cassettes can be opened by hand. 2. Storage Long term storage of up to 24 months store at 4 C or for 3 months at room temperature. For expiry date see box. 3. Sample and Running Buffer Preparation A. Sample Preparation We recommend using Optiblot LDS Sample Buffer 4X (ab119196) which has been specifically formulated for use with our gels. The ions in the sample buffer match the gel buffer and it has a higher density, making it compatible with the density of the running buffer. 3
Reagent Reduced Not Reduced Sample X µl X µl Water 13 - X µl 15 - X µl LDS Sample Buffer 4X (ab119196) 5 µl 5 µl DTT Reducer 10X (ab119199) 2 µl - Total Volume 20 µl 20 µl 1. Heat the samples, reduced or non-reduced, for 10 minutes at 70 C. 2. Reduced samples should be run within 2 hours to prevent re-oxidation. 3. Maximum volume that can be loaded in the wells is 35 µl. B. Running Buffer Preparation To enhance resolution our gels have been formulated with an improved ion system. Optiblot SDS Running Buffer formulation must be used with these gels. 4
Reagent Volume Optiblot 20X Run Buffer 40 ml Ultrapure water 760 ml Total Volume 800 ml Use Optiblot Reducing SDS Run Buffer (20X) (ab119195) for Reduced samples, and Optiblot SDS Run Buffer (20X) (ab119197) for non-reduced samples. To run reduced samples in ab119197 add 1 ml of Optiblot Antioxidant (800X) (ab119200) to the formulation above. We recommend using fresh buffer for each run for both the inner and outer chamber. Never use old buffers for the inner chamber (cathode). 4. Sample Loading Shortly before loading the samples, rinse the wells two times with ultrapure water. Use thin pipette tips to load samples near the bottom of the well. Optiblot Prestain Markers (ab119210), a prestained protein ladder is available. 5
5. Run Conditions Place the Optiblot gel cassette in the tank so that the shorter plate faces the buffer core. When running one gel, use a buffer dam to seal the other side. Fill the inner (cathode) chamber with 200 ml fresh running buffer until it overflows into the outer (anode) chamber. Check whether the cell has been assembled properly so that there are no leaks, then pour at least 400 ml running buffer into the outer chamber. Run the gel(s) until the blue dye front nears the bottom of the cassette as follows: Voltage 180V Start Current 90 ma/gel Ending Current 40 ma/gel Run Time 30 70 min 6
6. Gel Staining Remove the gel from the cassette into a staining tray and cover with 25 ml Optiblot Blue (ab119211). Protein bands will be visible within minutes. Leave the gel in stain for at least one hour before transferring into water, if you wish to dry or store the gel. Alternatively store the gel in stain. For silver staining, fix proteins for 10 minutes with a solution of 50% methanol, 10% acetic acid and 20mM sodium bisulfite. The sodium bisulfite can be added by diluting 1 ml of Optiblot Antioxidant (800X) (ab119200) in 200 ml fixative. Substitute this fix step with the manufacturer s silver staining protocol and follow the remaining manufacturer s method. Other gel stains can be used with Optiblot gels, please refer to protocols relevant to the specific stain. 7
7. Gel Drying The gels can be dried without cracking between cellophane after equilibrating with Optiblot Gel Drying Solution (ab119201). 1. Ensure that the gel has been staining for at least 1 hour. Further processing of the gel prior to completion of the staining process may result in protein destaining and reduced sensitivity. If this occurs simply restain the gel by incubating overnight in Optiblot Blue. 2. Submerse the gel in approximately 100 ml ultrapure water and incubate for at least 1 hour while gently rocking. Optionally adsorbent paper or paper towel can be added. Gels can be incubated overnight in water. 3. Incubate the gel in Optiblot Gel Drying Solution (ab119201) for 10 minutes and wet 2 cellophane membranes. 4. The gel is now ready for drying between the wetted cellophane membranes. 8
8. Gel Blotting Follow the general guidelines for your blotting unit. Optiblot TGS Blot Stock Buffer (10X/20X) (ab119198) contains 0.25M Tris (base), 1.92M Glycine, and 1% SDS. Dilute the Blot buffer: 10X for use in semi-dry blotters (SDB) 20X for other Tank Blotters (TB) Equilibrate gels in 1X Blot buffer for 5 to 10 minutes prior to blotting. Equilibrate pre-cut Nitrocellulose (NC) or PVDF membranes in 1X Blot buffer for 3-5 minutes. (PVDF must be wetted in 100% methanol or ethanol prior to equilibration in buffer.) 9
TB SDB Buffer Preparation PVDF NC PVDF NC 10x Blot Buffer (ml) 100 10 Methanol (ml) 200 400 10 20 Ultrapure water (ml) 1740 1540 82 72 Blotting Conditions TB SDB Voltage (V) 50 25 Blot time (hours) 2-4 0.5-1 Expected current (ma) 250 250-300 10
9. Buffer Recipes Optiblot Reducing SDS Run Buffer (20X) ab119195 0.6 M MOPS 1.2 M Tris 2% SDS 130 mm Sodium Bisulfite MOPS (free acid) Tris (free base) SDS Sodium Bisulfite 62.80 g 72.60 g 10.0 g 6.5 g Ultra pure water to 500 ml (~385 g) The ph should be between 8.2 and 8.3 @ 25 C. 11
Optiblot SDS Run Buffer (20X) ab119197 0.8 M Tricine 1.2 M Tris 2% SDS 50 mm Sodium Bisulfite Tricine (free base) Tris (free base) SDS Sodium Bisulfite 71.70 g 72.60 g 10.0 g 2.5 g Ultra pure water to 500 ml (~385 g) The ph should be between 8.2 and 8.3 @ 25 C. For Non-reduced samples (especially antibodies), omit the Sodium Bisulfite For best results use low conductance ingredients to formulate the buffers. Do NOT use acid or base to adjust ph of buffers. 12
Optiblot LDS Sample Buffer (4X) ab119196 40% Glycerol 4% LDS, 0.8 M Triethanolamine-Cl ph 7.6 4% Ficoll-400 0.025% Phenol Red 0.025% CoomassieBrilliant Blue G250 2 mm EDTA-2Na Glycerol Triethanolamine 6N HCl Lithium Dodecyl Sulfate Ficoll 400 EDTA Di-Sodium Brilliant Blue G250 Phenol red 4.0 g 1.2 g 0.93 g 0.40 g 0.40 g 0.007 g 0.0025 g 0.0025 g Ultra pure water to 10 ml (~4.5 g) 13
The ph should be between 7.7 and 7.8 @ 25 C. For best results use low conductance ingredients to formulate the buffers. Do NOT use acid or base to adjust ph of buffers. 14
Optiblot TGS Blot Stock Buffer (10X/20X) ab119198 1.92 M Glycine 0.25 M Tris 1% SDS Glycine 72.10g Tris (free base) SDS 15.20 g 5.0 g Ultra pure water to 500 ml (~385 g) The ph should be between 8.5 and 8.6 @ 25 C. For best results use low conductance ingredients to formulate the buffers. Do NOT use acid or base to adjust ph of buffers. Instructions: Prior to use, dilute this blot buffer stock solution: 10X for use in semi-dry blotters 20X for use in Tank Blotters 15
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