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Cleaver Scientific CVS10CBS, Инструкция по эксплуатации

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Cleaver Scientific CVS10CBS Скачать руководство пользователя страница 16

 

 

16 

 

Protein Blotting  

Setting up the blot sandwich  

N.B Remember to always wear gloves when dealing with proteins to avoid 
contaminations! 

1.

 

Following electrophoresis, remove the glass plate and transfer the gel 
into Transfer buffer. 

2.

 

Equilibrate the gel between 10-30min at room temperature on a shaker 
according to specific protocols. 

3.

 

Cut the membrane to the same size of the gel and equilibrate in 
Transfer buffer as well. 

 

NOTE: It is best to assemble the blotting sandwich in some transfer buffer to 
avoid the membrane to dry out and trapped bubble should be removed with 
a roller at each step during blotting sandwich assembly  

 

1.  Each blot sandwich should be set up according to the following order: 

-  Cassette clamp -ve (black) side placed in a tray or other suitable 

container 

-  Pre-soaked fibre pad. Note two can be used with thin gels.  

-  Two pieces of thick filter paper, about 2 

–

 3 mm thick, pre-soaked in 

buffer. 

-  Gel. 

-  Transfer membrane. Usually this requires pre-soaking but consult the 

manufacturer’s

 instructions for the type of membrane you are using. This 

should be smoothed so that no air bubbles have been trapped. 

-  Two pieces of thick filter paper, about 2 

–

 3 mm thick, pre-soaked in 

buffer. 

-  Pre-soaked fibre pad. Note two can be used with thin gels.  

-  Cassette clamp +ve (red). 

Содержание CVS10CBS

Страница 1: ...gue Numbers CVS10CBS SB10 Record the following for your records Model _____________________ Catalogue No _____________________ Date of Delivery _____________________ Warranty Period __________________...

Страница 2: ...e Guidance and restrictions 6 Setting up the Gel Tanks 6 Protein Electrophoresis 7 Casting Unit Preparation 7 Gel Preparation 9 Preparation of denatured protein samples for loading 11 Gel Pouring 11 G...

Страница 3: ...tly in position The unit should not be used if there is any sign of damage to the external tank or lid Acrylamide is a powerful neurotoxin in solution form Polymerized gels can contain some unpolymeri...

Страница 4: ...g List Checked by ________________________ Date ________________________ The packing lists should be referred to as soon as the units are received to ensure that all components have been included The...

Страница 5: ...de dishwashing liquid Hexane and Aliphatic hydrocarbons The units should not be left to in detergents for more than 30 minutes The units should never come into contact with the following cleaning agen...

Страница 6: ...ionally however a temporary conductivity caused by condensation must be expected Setting up the Gel Tanks Note Before setting up the Gel Tank please ensure that it has been properly cleaned and dried...

Страница 7: ...ause leaks Gel Cassette Assembly Assemble the glass plates in the CVS10DRIM casting running insert so that the bottom of the glass plates and the spacers are perfectly aligned on a flat levelled surfa...

Страница 8: ...When using the Slide Clamp Mini version simply slide both gates outwards until fully tightened When only one gel is being run the dummy plate must be used in the second position and fully tightened NO...

Страница 9: ...e clamps or screws as preferred by the user For those that prefer to use the screws rather than clamps the screws can be simply inserted into the screw holes The clamps can be removed by placing each...

Страница 10: ...olumes of solutions from the standard stock solutions These should be gently mixed avoiding generation of bubbles which will inhibit polymerization by removing free radicals Resolving Gel for 2 x 1mm...

Страница 11: ...combine the protein sample and 4 X sample buffer It is always advisable to use protein markers in one of the end lanes to indicate sizes of bands These should be prepared according to the manufacture...

Страница 12: ...n this use fresher stock solutions or add more APS and TEMED 7 Prepare the stacking gel solution 8 Before casting the stacking gel insert a piece of filter paper to dry the area in between the glass p...

Страница 13: ...ideways with the end s of the tank and pressed into the recess Or these can be fitted down the front of the tank Note NEVER FIT THESE UNDERNEATH THE MODULE IN THE BOTTOM OF THE TANK AS THIS WILL PREVE...

Страница 14: ...ve gel level Cooling packs are inserted behind the gels Outer Tank is filled to the maximum fill line Cooling is at a maximum 1000ml Gel Running 1 Fit the lid and connect to a power supply 2 Gels shou...

Страница 15: ...door Twist key applying pressure to both the clamping door and the CVS10D side cheek The door will now click open Repeat this process until you have opened both the doors 4 Remove the glass plates The...

Страница 16: ...and trapped bubble should be removed with a roller at each step during blotting sandwich assembly 1 Each blot sandwich should be set up according to the following order Cassette clamp ve black side pl...

Страница 17: ...entation and order the blot sandwiches were loaded in This can be done by noting which samples were loaded adjacent to each electrode and by marking the membrane with a pencil on one corner 2 Use of a...

Страница 18: ...he membrane is now ready to be probed Suggested Running Conditions please note that these are just guideline Duration of Blot SB10 One Hours Three Hours Overnight 16 hr 100V 125V 250mA 400mA 50V 200mA...

Страница 19: ...o 200ml with Distilled Water Stock 4 X Stacking Tris 0 5 M Tris HCL pH6 8 0 4 SDS To 110ml Distilled Water add 12 12 g of Tris base Add 8ml of 10 SDS Adjust pH to 6 8 with 1N HCl Add Distilled Water t...

Страница 20: ...03 g Bromophenol blue Aliquot into 1 5ml microcentrifuge tubes Store at 20 C Towbin Buffer 25mM Tris 192mM glycine 20 methanol pH8 3 Towbin Buffer SDS 25mM Tris 192mM glycine 20 methanol pH8 3 0 05 0...

Страница 21: ...pH will vary according to the freshness of the reagents used References 1 Sambrook Fritsch and Maniatis Molecular Cloning A Laboratory Manual Second Edition Cold Spring Harbor Laboratory Press 1989 2...

Страница 22: ...0 06 APS and 0 12 TEMED Gel does not polymerize Too little or too much APS or TEMED Failure to degas Temperature too low Poor quality acrylamide or bis Old APS Use 0 0 05 APS and 0 05 TEMED Degas mono...

Страница 23: ...rument manual Poor staining sensitivity Dirty staining trays Insufficient stain volume Insufficient staining time Reuse of staining solution Clean staining trays and other equipment with laboratory gl...

Страница 24: ...ing gel Uneven gel interface Remove salts from sample by dialysis or desalting column prior to sample preparation Use same buffer in samples as in gel Check buffer composition and dilution instruction...

Страница 25: ...extent that it prevents binding to nitrocellulose Alternatively reduce methanol concentration and switch to PVDF Protein precipitating in gel Use SDS in transfer buffer SDS can increase transfer effi...

Страница 26: ...brane PVDF Membrane is not completely wet Because of hydrophobicity of PVDF the membrane must be soaked entirely in methanol before equilibration in aqueous buffer Proteins might be transferring throu...

Страница 27: ...27 Too low background Increase antibody concentration protein sample concentration Consult manual included with antibody detection kit Total protein detection Consult stain or detection kit manual...

Страница 28: ...ormed by anyone other than CSL or an appointed distributor or representative are no longer under warranty from the time the unit was modified Units which have accessories or repaired parts not supplie...

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