NuPAGE Transfer Buffer (1x) Reagent: ... Na 3 VO 4 recipe: Aliquot 1 mL: Store at -20°C: 1) Prepare a solution of 200 mM sodium orthovanadate in ultrapure dH20 according to protocol by Gordon (1991) PubMed. Note: Solutions do not require degassing. *Add these last and mix well just before the gel is to be poured. Incubate the membrane protein-side up in the primary antibody solution with agitation, for 1 hour at room temperature or overnight at 2–8°C. Remove the blot from working solution and drain excess reagent. Prepare stacking gel solution according to the following table. Bis-Tris Transfer Buffer: 25 mM Bicine, 25 mM Bis-Tris (free base), 1 mM EDTA, pH 7.2. Dilute samples appropriately in NuPAGE sample buffer (for bis-tris) or Laemmli buffer (for TGX). No. ®NuPAGE MES [2-(N-morpholino) ethane sulfonic acid] SDS or MOPS [3-(N-morpholino) propane sulfonic acid] SDS Running Buffer for NuPAGE ® Novex ® Bis-Tris Gels ®NuPAGE Tris-Acetate SDS Running Buffer for NuPAGE ® Novex ® Tris-Acetate Gels ®NuPAGE ® Transfer Buffer for blotting of NuPAGE Novex Pre-Cast Gels . Run gel 200 V, 0.5 hour. Follow manufacture instructions for dry membrane preparations. The following recipes are for approximately 25 mL of separating gel, enough for four 1.0-mm thick mini gels. NuPAGE Antioxidant may be used with NuPAGE Transfer Buffer to enhance transfer of reduced proteins to membranes. NuPAGE Antioxidant may be used with NuPAGE Transfer Buffer to enhance transfer of reduced proteins to membranes. Prepare 400 ml NuPAGE transfer buffer (using 20x transfer buffer stock - kept at 4 deg) final concentrations in transfer buffer: 1.25X Transfer Buffer 5% Methanol Final volume 400 ml Note: depending on the protein properties, can use up to 15% Methanol. Product description NuPAGE™ Bis-Tris Gels are precast polyacrylamide gels designed for optimal separation *Optional but recommended because it makes it easy to form a good interface between the separating gel and the overlay. The specificity of the antibody-antigen interaction enables a target protein to be identified in the midst of a complex protein mixture. LC2676), Invitrogen NuPAGE LDS Sample Buffer (4X) (Cat. Here, you can find a collection of western blot recipes for commonly used protein electrophoresis and western blot buffers and stock solutions, and general western blotting protocols for chemiluminescent and fluorescent detection to guide you through your experiment. 37520), Pierce Blocker BSA (10X) in PBS (Cat. LDS Sample Buffer: 106 mM Tris HCl, 141 mM Tris Base, 2% LDS, 10% Glycerol, 0.51 mM EDTA, 0.22 mM SERVA Blue G250, 0.175 mM Phenol Red, pH 8.5. Remove gel from glass or plastic plates, cut off stacking gel. Ensure the volume of the antibody solution is enough to fully cover the membrane. when using high-performance substrates, such as SuperSignal substrates. No. No. Follow manufacture instructions for wet, semi-dry, or dry transfer. Impure methanol can increase transfer buffer conductivity and yield a poor transfer. Prepare the following stock solutions: all solutions can be stored at room temperature. For Research Use Only. If you are transferring 2 gels in the blot module, increase the methanol content to 20% to ensure efficient transfer of both gels. Scale volumes proportionally based on the number of gels to be cast. For a 100 mL solution, add 3.68 g Na3VO4 to 90 mL water and dissolve with stirring. Incubate the membrane with a sufficient volume of blocking buffer for 30–60 minutes at room temperature with agitation. ªÊ_ZËD§ªÌƒÚYw8öEuì¼`Q?ÂşT˜‘´[¯ª¶Ø(Å²n¼iÄ#¨àç-OÉ†¶�KadJÌ£’’Q�*ÇW¶â…¬[r¹r°Šl,æE!Kz-©ÕSâÿ©½Å²'V€;òo_0qÃ@‚emq³Æ»åßu‚k×�ÄUÛ+�»ş™o0ºÆÎõØ6dkãNĞëb˜‹ŒºC]Ú½=}ÿy‡GQ“ê0îœõƒø‹KE¡òÅÖ¥È…U¥f§×ldé)q9•KÚv:½~WnyÚpüöàrxúóêĞé5Ù¹Ò…(İ�‘5Ò&SœkSˆÜ-İ´P…¾�+r•Ê,—ÌÇÈÊâ#��Êfg#Ã¨ŠC|Z¢ÆÁ#Nƒî&§¤öp*Öh�Ÿà—®1ËsUÕª~´ ÀVÄK4=ËÒí€üş�€(~�€nĞ¤ègÊ÷}" ü/ø?ptñ„€=�§ÿ @üJ ÂM úş&. Check this using your samples. **Add these last and mix well just before the gel is to be poured. No. LC3675), NuPAGE Transfer Buffer (20X), 125 mL (Cat. Prepare transfer buffer for wet and semi-dry transfers based on gel chemistry. Konto erstellen, View recommended buffer formulations under Buffer Recipes tab. If you find this doesn’t work for your specific protein of interest, try our BlotBuilder Product Selection Tool to get a set of recommended products with a personalized western blot protocol. No. Dilute the primary antibody per supplier recommendations in the blocking buffer. Nitrocellulose or PVDF transfer membrane (e.g. No. No. Boil samples 5 minutes at 100ºC in heating block. Transferring Two Gels in One Blot Module . Directions for 1X Transfer Buffer: 1) Dissolve Tris base and glycine together in 1.6 L of ddH 2 O. Recommended secondary antibody dilutions to use with Thermo Scientific chemiluminescent substrates. 37525), Restore Western Blot Stripping Buffer, 500 mL (Cat. Not for use in diagnostic procedures. No. 6 No. Image the blot using film or appropriate imaging system. NuPAGE™ Transfer Buffer Wet transfer NP0006 Limited product warranty and licensing information Contents and storage Gel type Amount Storage NuPAGE™ Bis-Tris Gels Box of 2 or 10 gels Store at 4–25°C for up to 1 year. Tris-buffered saline with Tween 20 (TBST), Phosphate buffered saline with Tween 20 (PBST). NP0007), Novex Tris-Glycine SDS Running Buffer (10X), 500 mL (Cat. 10x Transfer buffer: For 4 L • 121.1 g Tris base • 576 g glycine • Bring up the volume to 4 L with ddH 2O 1x Transfer buffer: For 1 L • 700 mL cold ddH 2O • 100 mL 10x Transfer buffer • 200 mL methanol 20x TBS: For 4 L • 193.6 g Tris base • 640 g NaCl • Bring up the volume to 3.2 L with ddH 2O A western blot experiment, or western blotting, is a routine technique for protein analysis. Wash the membrane 3 times with agitation for 10 minutes each in wash buffer. No. 89900), Invitrogen Novex Tris-Glycine SDS Sample Buffer (2X) (Cat. No. No. NuPAGE™ Transfer Buffer Wet transfer NP0006 Limited product warranty and licensing information Contents and storage Gel type Amount Storage NuPAGE™ Bis-Tris Gels Box of 2 or 10 gels Store at 4–25°C for up to 1 year. No. LC2675), Novex Tris-Glycine Native Running Buffer (10X), 500 mL, 500 mL (Cat.
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