User Manual - Boca Scientific Laboratory Products
Contents
1. distilled water to100ml Transfer protocol wear gloves throughout the procedure OU OO Pre soak four pieces of blotting membrane to the size of the gel and equilibrate in transfer buffer If a PVDF membrane is used rinse it in methanol for 2 min before equilibration in the transfer buffer for 5 min Remove the gel from the cassette Carefully assemble the transfer stack on the anode Remove all air bubbles between the layers Air bubbles will affect the efficiency of the transfer process and may leave blank spots on the blotting membrane Transfer at room temperature at 0 8 mA cm of gel limit voltage to 15 V for 60 min When the transfer is complete turn off the power and remove layers until you reach the blotting membrane Remove the blotting membrane with a pair of forceps rinse in distilled water and air dry for 1 min Monitoring of protein transfer The efficiency of transfer can be monitored using prestained protein markers Alternatively the extent of protein transfer can be determined by staining the polyacrylamide gel after the transfer is complete or by staining the proteins directly on the blotting membrane Proteins on solid support can be stained with dyes such as India ink Amino Black or Coomassie Blue A recommended stain is Ponceau S which is reversible The detection limit is 1 to 2 micrograms Ponceau S staining for proteins on solid support 0 2 w v Glacial Acetic Acid 1 viv distilled water to2. 00m o Protocol 1 If the blotting membrane is dry re hydrate it with water or methanol if PVDF for 5 min 2 Stain the blotting membrane under constant shaking with the Ponceau S solution for 5 min 3 Destain the membrane under constant shaking with distilled water 4 If required wash the blot with 0 1N NaOH to remove the stain completely
3. Instruction Manual for IDGel from DGel Electrosystem Inc For complete protocol and product information visit www idgel com Running IDGel e Using IDGel in a Bio Rad Gel Running Apparatus Prior to inserting the gel in the apparatus Remove the gasket from the inner frame then turn the gasket around so that the flat side is facing outwards and re insert into the inner frame e Using IDGel in an Invitrogen Gel Running Apparatus Remove gel from packet and insert into the gel running apparatus Along the gel cassette slide the plastic insert enclosed in the IDGel box Close the chamber clamp according the manufacturer s instructions Loading and running IDGel Precast Gels e Dilute the samples to be analyzed with the appropriate volume of sample buffer in sealable micro centrifuge tubes e For protein electrophoresis heat the samples for 5 min at 100 C if denaturing conditions are used SDS PAGE e Load the samples e Run the gel s at an appropriate power supply setting see table below Buffer system Tris glycine SDS Laemmli Tris glycine native Tris Tricine SDS Tris acetate EDTA Voltage 150 VDC 250 VDC 125 VDC 125 VDC 200 VDC Approx per gel START 30 40 mA 55 65 mA 20 30 mA 45 55 mA 45 55 mA Approx per gel END 15 25 mA 30 40 mA 10 20 mA 20 30 mA 20 30 mA Current Wattage START 3 7 W 12 16 W 2 6 W 4 8 W 8 12 W Current wattage END 2 6 W 10 14 W 1 5 W 2 6 W 3 7 W Time 60 min 35 min 100 min 60 min 60 mi
4. _Ingredient_ Amount Final Stock Conc 14 4g 192 mM Methanol 100 200 ml 10 20 distilled water to 1 liter l 10 methanol is recommended when using PVDF blotting membranes while 20 is recommended when using nitrocellulose blotting membranes Transfer protocol RG DNDN Pre soak the porous pads and two pieces of filter paper cut to the size of the gel ex Whatman 3MM paper in transfer buffer for 5 min Cut a piece of blotting membrane to the size of the gel and equilibrate in transfer buffer If a PVDF membrane is used rinse in methanol for 2 min before equilibration in the transfer buffer for 5 min Remove the gel from the cassette and equilibrate in transfer buffer for 5 min Carefully assemble the transfer stack Remove all air bubbles between the layers If you do NOT do this then the air bubbles will affect the efficiency of the transfer process and may leave blank spots on the blotting membrane Place the stack into the tank unit making sure it is in the PROPER ORIENTATION Fill the chamber with transfer buffer Transfer at room temperature at 100 mA for 60 to 120 min When the transfer is complete turn off the power and remove the layers until you reach the blotting membrane Remove the blotting membrane with a pair of forceps rinse in distilled water and air dry for 1 min Transfer with a semi dry system Transfer buffer cool to 4C before use nount Final Stock Conc 192 mM Methanol 10 0 ml 10 v v
5. microliters 0 02 mM 37 formaldehyde 54 microliters 0 02 v v formaldehyde should be added just before use Stop solution Glacial Acetic Acid 5 ml 5 v v distilled water to 100 ml Protocol Note All steps of the following protocol should be performed under constant gentle shaking A sufficient amount of solutions should be used typically 100 ml is suggested in each step to cover the gel Fix in Gel Fixing solution 1 for 30 min Wash three times in Gel Fixing solution 2 each time for 10 min Treat with Sensitizer for 1 min Rinse twice with distilled water for 1 min each time Stain with Staining solution for 20 min Rinse twice with distilled water for 1 min each time Develop with Developing solution until the protein bands are readily visible generally 5 to 10 min avoid prolonged times as this will just increase the background staining Decant and replace with Stop solution for 10 min Store in distilled water EO LN og WESTERN BLOTTING Note The following protocols for Western blotting provide general information and conditions that may be applied to most transfer procedures However careful optimization should be considered if quantitative recovery of a particular protein is required since blotting efficiency is dependent on buffer composition blotting time and current membrane used etc Transfer with a tank blotting system Transfer buffer cool to 4C before use
6. n Attention IDGel is delivered without combs ready to use Note that due to the absence of a comb occasionally the well bottom may seem slightly curved or not flat This is due to cold temperature during packaging and this normal phenomenon will not affect the gel nor band migration In fact the wells will return to their normal flat position when the temperature increases or when buffer is added Use the gels immediately when removed from the pouch Opening the IDgel cassette e Hold the cassette with both hands with the notched plate facing you Press both thumbs onto the larger plate and at the same time pull both spacer extensions with another finger in the opposite direction e Completely separate the cassette halves by slowly pulling them apart with your fingers Once the gel is free from one of the cassette halves invert it carefully over the staining fixing or transfer solution Using a thin spatula or one of the spacers separate the gel from the cassette wall starting at one corner In general Maximum voltage 250 VDC The plastic is recyclable recycling code 1 The acrylamide matrix is colored to see the wells easily The color will migrate with the migration front It is non toxic and does not interfere with proteins or nucleic acids or with further analysis of your material Zymogram Western blot etc 2006 DGel Electrosystem Inc For research use only Not for use in clinical diagnostics Patent pending Guide t
7. o protein staining procedures Coomassie Blue R 250 staining of proteins Staining solution Brilliant Blue R 250 1g 0 1 w v Methanol 500 ml 50 v v Glacial Acetic Acid 100 ml 10 v v Distilled water Ju To 1 liter Destaining solution ail ule Methanol 250 ml 5 v v Glacial Acetic Acid 80 ml 8 v v distilled water to 1 liter completely dissolve the dye in methanol and then add the other components Protocol e Place the gel constant shaking in 100 ml of Staining solution for 20 min e Wash the gel with 100 ml distilled water for 10 seconds e Destain the gel under constant shaking in 200 ml of Destaining solution with shaking for 30 min e Repeat step 3 with fresh Destaining solution until the background is clear generally 3 X 30 min however time will vary with gel conc Silver staining of proteins Place the gel in distilled water Gel fixing solution1 Ethanol 50 ml 50 Glacial Acetic Acid 10 ml 10 v v distilled water to 100 ml Gel fixing solution2 Ethanol 90 ml 30 v v distilled water to 300 ml 200 mM Na thiosulfate 400 microliters 0 8 mM distilled water to 100 ml Staining solution Silver nitrate 0 2g 11 8 mM 37 formaldehyde 54 microliters 0 02 v v distilled water to 100 ml formaldehyde should be added just before use Developing solution Na carbonate 6 0g 566 mM 200 mM Na thiosulfate 10
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