Fastblot User Manual

Fastblot User Manual
Fastblot
B31
B32
B33
B34
B43
B44
Code-No. 014-800
Code-No. 014-000
Code-No. 014-100
Code-No. 014-200
Code-No. 015-100
Code-No. 015-200
Manual
January 2001
!! Warning !!
Please read these instructions carefully
before using this apparatus!
Biometra biomedizinische Analytik GmbH
Rudolf-Wissell-Str. 30
D-37079 Göttingen
Postfach 1544
D-37005 Göttingen
Tel: ++49 – (0)5 51 / 50 68 6-0
Fax: ++49 – (0)5 51 / 50 68 6-66
e-mail: [email protected]
internet: http://www.biometra.de
Service Department
Rudolf-Wissell-Str. 14-16
D-37079 Göttingen
Tel.: ++49 – (0)5 51 / 50 88 1-10 or -12
Fax.: ++49 – (0)5 51 / 50 88 1-11
e-mail: [email protected]
Contents:
1 Intended Uses And Specifications.......................................................................................... 2
2 Safety...................................................................................................................................... 4
3 Set Up..................................................................................................................................... 5
3.1 Unpack And Check .......................................................................................................... 5
3.2 Electricity Supply............................................................................................................. 5
3.3 Important Electrical Safety Notes.................................................................................... 5
4 Location.................................................................................................................................. 5
5 Additional Comments ............................................................................................................. 6
5.1 Efficiency Of The Transfer .............................................................................................. 6
5.2 Blotting Conditions.......................................................................................................... 6
5.3 Cooling............................................................................................................................. 7
6 Handling ................................................................................................................................. 8
7 Discontinuous Buffer System............................................................................................... 11
8 Transfer Of DNA (Agarose Gels) ........................................................................................ 13
9 Solutions............................................................................................................................... 14
10 Service................................................................................................................................ 16
11 Notes................................................................................................................................... 18
1
1 Intended Uses and Specifications
The Biometra Fastblot is intended to be used for electroblotting. Electroblotting has become
an important method transfering proteins from polyacrylamide gels onto nitrocellulose, nylon
or other membranes.¹,²,³
The Fastblot is available in two different sizes: 12 x 12 cm and 16 x 20 cm electrode surface.
Both sizes are available with or without internal cooling.
Conventional blotting (tank blotting) is performed in a big buffer chamber and the procedure
is very buffer and time consuming. The more recent method of semi-dry blotting between
carbon plate electrodes allows a more rapid and homogeneous transfer.4
Pure (unmodified) carbon plates originally used for semi-dry blotting show extensive
corrosion and decomposition. The electrodes used for the Fastblots are made of special
modified carbon based material. This material is extremely resistant and allows transfer at
higher currents. Transfers are usually completed within 10 to 30 minutes and are uniform in
all areas of the polyacrylamide gel. With the semi-dry blotting procedure even high molecular
weight proteins (> 100 kDa) are transferred from (SDS-)polyacrylamide gels to a membrane
at high currents.
Warming up of the apparatus and blot-sandwich during the transfer at high current conditions
or during blotting of native proteins can be compensated by the internal cooling system of the
Fastblot B31 or B33.
References:
1. Towbin, H., Staehlin, T. and Gordon, J. (1979); Proc. Nat. Acad. Sci. 76, 4350 - 4356
2. Bittner, M., Kupferer, P. and Morris, C.F. (1980); Anal. Biochem. 102, 459 - 571
3. Burnette, W.N. (1981); Anal. Biochem. 112, 195 - 203
4. Kyse-Andersen, J. (1984); J. Biochem. Biophys. Meth. 10, 203 - 209
2
The Fastblot comes complete with:
- Body with electrode (anode) - B31, B33, B43: including cooling device
- Lid with electrode (cathode)
- Wires (permanently attached to the lid) with safety connectors
- Warranty Registration card
- Manual
Systems and Accessories:
Systems:
014-800
014-000
014-100
014-200
015-100
015-200
Fastblot B31, complete system, electrode surface 12 x 12 cm,
with flow-through cooling, incl. manual
Fastblot B32, complete system, electrode surface 12 x 12 cm,
without cooling, incl. manual
Fastblot B33, complete system, electrode surface 16 x 20 cm,
with flow-through cooling, incl. manual
Fastblot B34, complete system, electrode surface 16 x 20 cm,
without cooling, incl. manual
Fastblot B43, complete system with special metal electrodes,
electrode surface 16 x 20 cm, with flow-through cooling, incl. manual
Fastblot B44, complete system with special metal electrodes,
electrode surface 16 x 20 cm, without cooling, incl. manual
Accessories:
014-001
092-001
092-002
092-003
3030 931
Cooling cushion for B33/B34
Blotting paper, 21 x 29.7 cm, 190 g/qm, 0.35 mm thickness, 25 pcs.
Blotting paper, 21 x 29.7 cm, 330 g/qm, 0.90 mm thickness, 25 pcs.
Blotting paper, 10.5 x 13 cm, 700 g/qm, 1.30 mm thickness, 20 pcs.
Whatman 3MM, 58 x 68 cm, 185 g/qm, 0.34 mm thickness, 100 pcs.
3
2 Safety
Do not operate this equipment if buffer or water leaks from the Fastblot.
Do not operate this equipment if cracks are present in the body or the
safety cover of the Fastblot.
Do not operate this equipment if the electrical connection cables are worn
or frayed.
Danger! High voltage! Disconnect the unit from the Power Supply before
opening the safety lid. Never try to detach the cables from the lid and
never open the system during blotting!
The maximum power for B31/B32 is 5 Watts (W) and for the
B33/B34/B43/B44 is 10 Watts (W)
Using the cooling option do not mix up the "in" and "out" plugs for the
cooling water. The connection for the water cooling with the smaller
diameter is the inlet the connection with the bigger diameter is the outlet.
The cooling water flow should be 0.5 - 1.0 l / min (max.).
Best cooling is obtained using a refrigerated circulator (chiller)
(Code-No. 043-200 or 043-290) with a temperature of 5°C.
(Attention: Reduce flow rate to max. 1 l/min and use no organic solvents
or alcohol !)
Do not use alcohol (e.g. methanol, ethanol) or organic solvents for cooling
or cleaning the gel apparatus.
Do not heat the apparatus over 50°C.
Do not use alkaline transfer buffers pH > 10 !
Do not change polarity of the electrodes: The anode (+) is on the body of
the Fastblot and the cathode (-) is fixed in the lid !
4
3 Set Up
3.1 Unpack and Check
Unpack and carefully examine the Fastblot. Report any damage to BIOMETRA. Do not
attempt to operate this device if physical damage is present. Save all packing material if
damage is found.
!! Attention !!
Please fill out and send back the warranty registration card. This is
important for you to claim full warranty.
3.2 Electricity Supply
The Fastblot has been designed to operate with D.C. current.
Warning: The Fastblot must not be earthed.
3.3 Important Electrical Safety Notes
Do not operate this equipment if any of the following conditions exists:
- If buffer or water leaks from the Fastblot.
- If cracks are present in the body or the safety cover of the Fastblot.
- If the electrical connection cables are worn or frayed.
4 Location
Place the chamber in proximity to the Power Pack with which it is to be connected. Be sure to
place the chamber in a safe, dry location away from the edge of the working surface.
5
5 Additional Comments
5.1 Efficiency of the Transfer
The efficiency of the transfer can be checked by applying Coomassie blue prestained standard
proteins on the gel.
After the blotting it is possible to stain the gel with Coomassie blue to check the completeness
of transfer.
5.2 Blotting Conditions
The blotting conditions (time, current) should be optimised for every protein. Time and
transfer rates of different proteins are summarised in Figure 1.
Using the plot shown in Fig. 1 it is possible to carry out a rough estimation of the transfer
times.
• The transfer time is also influenced by the thickness and the acrylamide concentration of
the gel.
• If the transfer time is too long the protein may pass through the blotting membrane and is
lost. You can check this by adding another layer of membrane and check the second
membrane also for protein after blotting.
Fig.1: Electrophoretic transfer of proteins.
SDS-PAGE, Acrylamide concentration: 10%; Nitrocellulose blotting membrane, pore
size 0.45 µm; current: 5 mA per cm2 gel; thickness of gel: 1.0 mm; transfer buffer:
Tris/Glycine/SDS
Using B43/B44 can reduce the transfer time in the range of 20%.
6
5.3 Cooling
When blotting high molecular weight proteins (> 100 kDa) blotting will take up to 30 min. In
this case or when blotting native proteins/enzymes, we suggest using the cooling system of
the Fastblot B31 or B33.
7
6 Handling
Step 1:
After finishing the electrophoresis immediately remove the gel from the glass plates and cut
out from the gel the part you want to blot.
Attention: Wear gloves !
Step 2:
Cut the blotting paper to the gel size and soak five (5) layers of 0.35 mm thick Blotting paper
(Code-No. 092-001) or three (3) layers of 0.90 mm thick Blotting paper (Code-No. 092-002)
or two (2) layers of 1.3 mm thick Blotting paper (Code-No. 092-004) with transfer buffer.
Place the soaked filter paper on the anode (+). The anode is the plate electrode on the body of
the Fastblot.
Step 3:
Cut the membrane (usually nitrocellulose) carefully to gel size, soak it also with buffer and
place it on the filter paper prepared in step 2.
Attention: Avoid the inclusion of air bubbles (“white spots“ on the
membrane) and wear gloves when handling the membrane !
Step 4:
Place the SDS-polyacrylamide gel on top of the membrane.
Attention: Avoid air bubbles !
Step 5:
Add five (5) layers of 0.35 mm thick Blotting paper (Code-No. 092-001) or three (3) layers of
0.90 mm thick Blotting paper (Code-No. 092-002) or two (2) layers of 1.3 mm thick Blotting
paper (Code-No. 092-004) previously soaked with transfer buffer on top of the
polyacrylamide gel.
DO NOT use alkaline transfer buffers pH > 10 !
Step 6:
Remove air bubbles which might be included by carefully rolling out the blot-sandwich with
a tube or a pipette.
8
Step 7:
Connect the lid with the body of the Fastblot. Take care that lid and body are attached
completely horizontally. When handling thicker gels put a weight of 1 - 2 kg on top of the lid
(e.g. a beaker glass with cold water).
Step 8:
If you have assembled the “sandwich“ for blotting, the set up should look as shown in Fig. 2.
Fig. 2:
Set up of filter paper (blotting
paper), gel and blotting
membrane between the plate
electrodes. Blotting paper and
blotting membrane are used
in the same size as the gel.
Step 9:
Switch on your Power Pack, e.g. Biometra Standard Power Pack P25 (Code-No. 040-800) or
one of the Biometra High Voltage Power Packs at a constant current of 0.8 – 1.0 mA per cm2
2
of gel (max. 5 mA per cm of gel).
Attention: The maximum power for B31/B32 is 5 Watts (W) and for
the B33/B34/B43/B44 is 10 Watts (W).
The electrodes consist of different materials. DO NOT change
polarity of the electrodes: The anode (+) is on the body of the Fastblot
and the cathode (-) is fixed on the lid !
Step 10:
If the lid is getting warm during the blot procedure reduce the current or add a pre-cooled
cooling cushion (Code-No. 014-001).
Step 11:
If you are using the water cooling, please note the following:
The connection for the water cooling with the smaller diameter is the inlet, the
connection with the bigger diameter is the outlet.
Attention: Never mix up inlet and outlet !
The cooling water flow should be 0.5 - 1.0 l / min (max.).
Use no organic solvents or alcohol !
9
Best cooling is obtained using a refrigerated circulator (chiller) (Code-No. 043200 or 043-290) with a temperature of 5°C. (Attention: Reduce flow rate to max.
1 l/min)
Step 12:
After finishing the blot transfer switch off the current first, disconnect Power Pack and
Fastblot and turn off the cooling water. After this take off the lid!
Danger! High voltage! Never try to detach the cables from the lid and
never open the system during blotting!
Step 13:
Remove the blot-sandwich carefully and take the membrane out of the “sandwich”. Then
proceed with staining or immunoassay.
Never touch the membrane with fingers (without gloves)!
Step 14:
After every blotting procedure clean the plate electrodes with distilled water. A paper
towel can be used for drying.
10
7 Discontinuous Buffer System
The use of discontinuous buffer systems allows a higher resolution of the proteins and the
transfer kinetics can be improved. The method essentially resembles the blotting with
continuous buffer system. The main difference is that 3 different buffers are used.
Step 1:
After the electrophoresis incubate the gel in cathode buffer for five minutes.
Step 2:
Moisten the anode with anode buffer II.
Step 3:
Adapt 6 layers of the 0.90 mm of thick blotting paper (Code No. 092-002) to the size of the
gel.
Step 4:
Soak 2 layers of the blotting paper in anode buffer II and place them on the anode.
Step 5:
A further layer of the adapted blotting paper is soaked with the anode buffer I and added to
the sandwich.
Step 6:
An PVDF membrane (Code No. 231-578) cut to the size of the gel is successively incubated
in methanol (1 – 2 seconds), in H2O (5 minutes) and in anode buffer II. Subsequently, it is
placed on the transfer sandwich.
Step 7:
The SDS-Polyacrylamide gel is put on top of the membrane.
Step 8:
Three layers of blotting paper are soaked in cathode buffer and placed on top of the gel.
Note:
Avoid the inclusion of air bubbles in the blotting sandwich
and wear gloves!
Step 9:
Connect the lid with the body of the Fastblot. Take care that lid and body are attached
completely horizontally.
Step 10:
If you have assembled the “sandwich“ for blotting, the set up should look as shown in Fig. 3.
11
Fig. 3: Set up of filter paper (blotting paper), gel and blotting membrane between the plate
electrodes. Blotting paper and blotting membrane are used in the same size as the gel.
For the further proceedings please refer to chapter ”6 Handling”.
12
8 Transfer of DNA (Agarose Gels)
Using the Fastblot also nucleic acids can be transferred to membranes.
(Nevertheless the use of a Vac-Blot System (Code No. 053-000 and 053-300) is
recommended.)
Proceeding:
Prior to the transfer, the DNA should be pre-treated in order to increase the efficiency of the
blotting.
0.25 M HCl
7 min
Depurination
Denaturation
0.5 M NaOH
1.5 M NaCl
15 min
Neutralisation
3.0 M NaCl
0.5 M Tris, pH 7.4
15 min
Build up the blotting sandwich as described in chapter 6 Handling, but use 1 x TAE or
1 x TBE as transfer buffer and 10 layers of blotting paper (Whatman 3MM Chr paper, Code
No. 3030700, 3030704, 3030861, 3030917 and 3030931) on either side of the membrane/gelsandwich. For the transfer of DNA, the nylon membrane (Code No. 231-576) has to be on the
anode side of the gel. Using 3 – 5 mA per cm² of gel the transfer takes about 30 minutes.
Do not use nitrocellulose membranes, because they are not stable in basic
solutions!
13
9 Solutions
Transfer buffer, pH 8.3
25 mM Tris-base
150 mM Glycine
10 % Methanol
Optional:
Dilute the electrophoresis running buffer with one or two volumes of distilled water.
Running buffer (Laemmli System):
25 mM Tris-base
192 mM Glycine
0.1 % SDS (Sodiumdodecylsulphate)
For blotting dilute with distilled water!
Staining Solution (1 litre)
2.0 g Coomassie Brilliant Blue R 250
0.5 g Coomassie Brilliant Blue G 250
425 ml Ethanol
50 ml Methanol
100 ml Acetic acid
425 ml bidest. water
Stir overnight; filtrate before use; store in dark bottle!
Destaining solutions (acrylamide gels)
Fast destaining in:
45 % Ethanol
10 % Acetic acid
45 % bidest. water
Slow destaining in:
25 % Isopropanol
10 % Glacial acid
65 % bidest. water
Final destaining in:
7 % Acetic acid in bidest. water
14
Buffers for discontiuous buffer system:
Anode buffer I:
300 mM
Tris/HCl, pH 10,4
20 % (v/v) Methanol
Anode buffer II:
25 mM
Tris/HCl, pH 10,4
20 % (v/v) Methanol
Cathode buffer:
25 mM
Tris/HCl, pH 9,4
40 mM
Capronic acid
20 % (v/v) Methanol
Buffers for DNA transfer
50 x TAE-Buffer-Stock (1 Liter):
242
g
Tris base, pH 8,0
57,1 ml
Glacial acetic acid
100 ml
EDTA (0,5 M)
5 x TBE-Buffer-Stock (1 Liter):
54
g
Tris base, pH 8,0
27,5
g
Boric acid
20 ml
EDTA (0,5 M)
15
10 Service
Should you have any problems with this unit, please contact our service department or your
local Biometra dealer:
Biometra biomedizinische Analytik GmbH
Service Department
Rudolf-Wissell-Straße 14 - 16
D-37079 Göttingen
Phone:++49 – (0)5 51 / 50 88 1- 10 or 12
Fax: ++49 – (0)5 51 / 50 88 1 –11
e-mail: [email protected]
If you would like to send the unit back to us, please read the following return
instructions.
Instructions for return shipment
• Return only defective devices. For technical problems which are not definitively
recognisable as device faults please contact the Technical Service Department at
Biometra.
• Use the original box or a similarly sturdy one.
• Label the outside of the box with “CAUTION! SENSITIVE INSTRUMENT!”
• Please enclose a precise description of the fault, which also reveals during which
procedures the fault occurred, if possible.
• Important: Clean all parts of the instrument from residues, and of biologically dangerous,
chemical and radioactive contaminants. Please include a written
confirmation (use the “Equipment Decontamination Declaration” following
on the next page) that the device is free of biologically dangerous and
radioactive contaminants in each shipment. If the device is contaminated, it is
possible that Biometra will be forced to refuse to accept the device.
• The sender of the repair order will be held liable for possible damages resulting from
insufficient decontamination of the device.
• Please enclose a note which contains the following:
a) Sender’s name and address,
b) Name of a contact person for further inquiries with telephone number.
16
Equipment Decontamination Certificate
To enable us to comply with german law (i.e. §71 StrlSchV, §17 GefStoffV and §19 ChemG) and to avoid
exposure to hazardous materials during handling or repair, will you please complete this form, prior to the
equipment leaving your laboratory
COMPANY / INSTITUTE __________________________________________________________________
ADDRESS _______________________________________________________________________________
TEL NO _________________________________
FAX NO _______________________________
E-MAIL _________________________________________________________________________________
EQUIPMENT
If on loan / evaluation
Model
Serial No
______________
__________________
______________
__________________
______________
__________________
______________
__________________
Start Date: ________
Finish Date ________
Hazardous materials used with this equipment
_________________________________________________________________________________________
_________________________________________________________________________________________
_________________________________________________________________________________________
Has the equipment been cleaned and decontaminated? YES / NO (delete)
Method of cleaning / decontamination
_________________________________________________________________________________________
_________________________________________________________________________________________
_________________________________________________________________________________________
NAME ________________________________
POSITION ______________________________
(HEAD OF DIV./ DEP./ INSTITUTE / COMPANY)
SIGNED ______________________________
DATE __________________________________
PLEASE RETURN THIS FORM TO BIOMETRA GMBH OR YOUR LOCAL BIOMETRA DISTRIBUTOR
TOGETHER WITH THE EQUIPMENT.
PLEASE ATTACH THIS CERTIFICATE OUTSIDE THE PACKAGING.
INSTRUMENTS WITHOUT THIS CERTIFICATE ATTACHED WILL BE
RETURNED TO SENDER.
17
11 Notes
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18
Warranty
This laboratory instrument is produced with the highest practical standards of materials,
workmanship, and design. The design and manufacture of parts have been conceived with one
purpose - to produce units which will give satisfactory service.
Biometra GmbH guarantees this unit to be free from defects in materials or workmanship
under normal use or service for 12 month from date of shipment. If, during this time, this unit
proves defective in materials or workmanship, Biometra GmbH will repair or replace it free
of charge if returned to us prepaid. This guarantee does not cover damage in transit, damage
caused by carelessness, misuse or neglect, or unsatisfactory performance as a result of
conditions beyond our control; or consequential losses as a result of failure of our product.
___________________________________________________________________________
Biometra biomedizinische Analytik GmbH
Rudolf-Wissell-Str. 30
D-37079 Göttingen
Postfach 1544
D-37005 Göttingen
Tel: ++49 – (0)5 51 / 50 68 6-0
Fax: ++49 – (0)5 51 / 50 68 6-66
e-mail: [email protected]
internet: http://www.biometra.de
Service Department
Rudolf-Wissell-Str. 14-16
D-37079 Göttingen
Tel.: ++49 – (0)5 51 / 50 88 1-10 or -12
Fax.: ++49 – (0)5 51 / 50 88 1-11
e-mail: [email protected]
19
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