Ibi IB44000 Manuel utilisateur

MANUAL

OPERATION

Semi-Dry Blotters

16cm x 16cm | 24cm x 30cm

IB44000 | IB45000

Important Safety Information!

1 Please read this manual carefully before operating your new IBI Semi-Dry Blotter.

1 This manual contains important operating and safety information.

1 To best use this product, please read the entire manual carefully prior to use.

1 To avoid possible injury, this product should only be used for its intended purpose.

Package Contents | Unpacking

Upon receiving this product, please verify all of the noted parts and accessories are contained in this package. Awareness of the stated cau-

tions and warnings contained within this manual, compliance with recommended operating parameters, and maintenance requirements are

important for safe and satisfactory operation.

1 Semi-Dry Blotter; based on size IB44000 or IB45000

1 Top cathode (black) and bottom anode (Gray) assemblies.

1 Power Cord Set - Black & Red (To connect Semi-Dry Blotter to Power Supply)

1 Operation Manual

Save all packing material, and these instructions, if the Semi-Dry Blotter is received damaged. This shaker was carefully packaged and thor-

oughly inspected before leaving the factory. Responsibility for its safe delivery was assumed by the carrier upon acceptance of the shipment;

therefore, claims for loss or damage sustained in transit must be made with the carrier.

Note: Carefully inspect all items in the package to insure no items are broken or missing. If there are items broken, please inspect

the package carefully for signs of shipping damage. If there is ANY sign of shipping damage, please contact the carrier and le a

claim with them immediately. Contact the distributor from which you purchased the item or IBI Scientic directly for assistance

at (800) 253-4942 or (563) 690-0484.

Product Specications

IB44000

Unit Dimensions: 9.25”(l) x 9.25”(w) x 5.25”(h)

Working Surface Dimensions: 7” [16cm] x 7” [16cm]

Weight: 3.75lbs [1.7Kg]

Power Cords (Red & Black) 36” & 36”

Recommended Trans Units: 1 - 6

IB45000

Unit Dimensions: 14.25”(l) x 12”(w) x 3”(h)

Working Surface Dimensions: 9.5” [24cm] x 11.8” [30cm]

Weight: 9.5lbs [4.3Kg]

Power Cords (Red & Black) 36” & 36”

Recommended Trans Units: 1 - 3

Product Set-up::

The diagram shown below shows how to set up your new Semi-Dry Blotter. Make certain you do NOT have the power cords connected to the

DC power supply until you are ready to proceed with an actual transfer.

Principles of Operation

Mixtures of biomedical products can be separated on the basis of their molecular sizes and ionic characteristics by polyacrylamide or

agarose gel electrophoresis. Dierent proteins will appear as bands on the gels after staining. In order to further identify the characteristics

of these proteins, components can be transferred and immobilized on a solid support in order that further detection can be achieved using

radioisotopic or immunological methods. The transfer of these components from gels to a solid support requires either an electrical current,

vacuum suction or diusion to move the components from the gel to the solid support. The IBI Semi-Dry Blotting apparatus is a device that

can migrate these components from the gel to a solid support by way of electrical current.

The fastest method for the transfer of proteins, RNA, and DNA from a gel to a solid support is electrophoretic transfer. To do this, the gel

is layered in contact with the membrane sandwiched between several layers of lter paper soaked with buer solutions forming a stack or

single trans unit. A constant current is applied perpendicular to the gel and lter papers causing the sample to migrate from the gel on to

the membrane. The membrane is then ready for staining or other developmental methods utilizing a PTS instrument, autoradiography, or

enzymatic methods.

The IBI Semi-Dry Blotting apparatus utilizes a semi-dry electrophoretic method for transferring molecules of interest from gels to solid

platforms/membranes. This method is safer and considerably more ecient and economical than wet tank methods. It can also be used for

transfer of proteins from isoelectrofocused gels or slab gels.

The IBI Semi-Dry Apparatus

The IBI Semi-Dry apparatus consists of a specially designed, graphite covered, cathode plate and anode plate. The cathode plate is part of

the lid assembly (black) and the anode plate is part of the base assembly (gray). A Trans-Unit stack is formed by the gel and the membrane

being in contact and sandwiched between lter papers soaked in buer solution.

An electric current is applied perpendicular to the gel and lter papers. The cathode paper is saturated with the cathode buer and the anode

paper is soaked with anode buer. In electrotransblotting, a pH gradient between the cathode and anode side of the trans unit is established

so that negatively charged molecules can migrate toward the positively charged side of the trans unit. The bands from the gels migrate out of

the gels and bind to the nitrocellulose membrane (or other membrane supports depending on which type of blotting is being accomplished).

Once the bands reach the membrane they are immobilized until the portion of the membrane is saturated with the migrated molecules.

Excess unbound molecules will migrate into the anode lter paper until it reaches the dialysis membrane which will block further migration of

the molecules into the next trans unit. Several trans units can be stacked on top of each other in order to increase the throughput of the appa-

ratus. The IB44000 Semi-Dry unit is designed to accommodate as many as 6 trans units stacked on top of each other and run simultaneously.

The IB45000 Semi-Dry unit is designed to run 2 – 3 trans units simultaneously.

The graphite covered cathode and anode serve as the electrodes for the electrophoretic transfer and are designed to dissipate heat during

the process. The time needed to transfer bands from various types of gels to membranes using various current densities are listed in Table 1.

Table 1. Recommended current settings for electro-transblotting proteins

Current Densit Trans Units Time Limit

0.8mA/cm2 1 – 6 1 – 2 hours

2.5mA/cm2 1 – 6 30 – 45 minutes

4.0mA/cm2 1 – 6 10 – 30 minutes

Building a Trans Unit Stack

Items not included but required to construct a Trans Unit:

• Polyacrylamide or Agarose gel that has been through electrophoresis.

• IB95011 - Thick Blot paper for absorbing cathode or anode buer – cut 10cm x 10cm – (pack of 10)

• IB95020 - 0.2mm Nitrocellulose Membrane cut 10cm x 10cm. (pack of 10 membranes)

• IB95031 - 0.45mm PVDF membrane cut 10cm x 10cm. (pack of 10)

• IB95050 - Filter pads for inserting between trans units.

If you are running larger gels you will need to purchase the above items from another supplier as we work to provide trans unit materials for larger gels.

IBI Power supply:

• SH-500XL - 500v / 500mA power supply with 4 outputs

• SH-300XL - 300v / 400mA power supply with 4 outputs

Buer Solutions for SDS PAGE Proteins:

• Anode Buer Solution #1: 0.3M TRIS, 20% methanol, pH 10.4

• Anode Buer Solution #2: 0.025M TRIS, 20% methanol, pH 10.4

• Cathode Buer Solution: 0.025M TRIS/0.04M amino caproic acid, 20% methanol, pH 9.4

• IBI TRIS Powder: IB70142(500gm), IB70144(1Kg), IB70145(5Kg)

Operational Procedures and

Techniques for Western Blotting

Warning: Be sure to wear gloves, safety glasses and lab coat/apparel when working with gels, buers and semi-dry apparatus.

Trans Unit Assembly:

• Pre-soak the gel in anode buer for 2 to 10 minutes at room temperature.

• Pre-cut blotting/lter paper and the solid support membrane to the same size as the gel.

• Pre-soak the membrane in distilled water at room temperature. If membrane is PVDF, soak in methanol.

• Place 2 sheets of thick blot paper/lter paper, saturated with anode buer #1 on to the anode plate (base plate) of the semi-dry device.

Now place an additional blot paper soaked with anode buer #2 on top of the other two blot papers. (see recipes for anode buer #1

and #2 above)

• Lay the wet membrane from step 3 on top of the blot papers.

• Carefully lay the pre-soaked gel on top of the membrane making certain not to have any air bubbles between the gel and the membrane.

• Place a thick blot paper soaked in cathode buer on top of the gel.

• If stacking another trans unit on top, lay a dialysis membrane or IB95050 ne lter pad, pre-soaked in distilled water and cut to the

same size as the blot paper, on top of the cathode blot paper and proceed to build another stack of trans unit.

• When you have reached the top of the nal trans unit stack, place two blot papers on top soaked in cathode buer.

• Place the cathode plate cover (Black) on the assembled trans unit stack. Be sure to align the anode relief slot.

• Connect the red anode lead wire to the red jack on the semi-dry apparatus. Connect the black cathode lead wire to the black jack on the

base of the semi-dry apparatus.

• Connect the anode and cathode lead wires to the corresponding jacks on the DC power supply.

• Turn on the power supply and set the unit for constant current. Adjust the current setting on the power supply to match the amperage

requirements as determined by Table 1.

• When transfer is completed: TURN OFF power supply and unplug power cords.

• Remove the cathode cover plate

• Carefully peel o the blot/lter paper and discard

• Carefully peel o the gel which can now be stained or discarded.

• Peel o the membrane with forceps and ready for xation and post transblotted development process.

Operational Procedures and

Techniques for Semi-Dry DNA Blotting

If the user is not using an automated developing device and is developing blots and gels by a manual method, the following is an overview of

suggested solutions and methods. NOTE: The lter pads and blotting paper noted below are 10cm x 10cm. If you need larger you will need

to source elsewhere.

• DNA is separated on an IBI Molecular Biology agarose gel (0.7 – 1%) poured with 1X TBE buer or IBI Electrophast buer. For genomic

DNA use 0.7% and for plasmid digest use 0.8% - 1%

• After electrophoresis trim away non-essential portions of the gel. Prepare 2 buer pads (IB95050 Fine Filter pads – 10cm x 10cm)) and

2 thick blot papers (IB95011 – 10cm x 10cm) and one charged modied nylon membrane cut to the exact size of the trimmed gel.

• Soak gel twice in 2 gel volumes of DI water (IB42110 – 500ml Molecular Biology Grade Water) for 10 minutes.

• Soak gel twice in two gel volumes of denaturing solution #1 for 30 minutes at room temp with constant shaking.

• Soak gel twice in two gel volumes of neutralizing solution #2 for 30 minutes at room temp with constant shaking.

• Soak gel twice in two gel volumes of solution #3 – 1X TBE for 30 minutes. (IB70150 – 10X TBE buer in 1L)

• Wet one blotter lter pad and one thick blot paper in solution #3 (1X TBE). Place the lter pad on the anode plate and press down to

minimize bubbles. Place the thick blot paper, soaked in 1X TBE on top of the lter pad. Roll out to eliminate bubbles

• Equilibrate the charge modied nylon transfer membrane in solution #3. 1X TBE, for at least 10 minutes and place it on top of the thick

blot paper and eliminate air bubbles. Do not move the gel once it is in place.

• Carefully place the equilibrated gel on top of the membrane making certain to eliminate air bubbles.

• Wet a second blotter lter pad and thick blot paper in solution #3, 1X TBE. Place the thick blot paper on top of the gel then place the lter

pad on top of the blot paper, eliminate air bubbles.

• Carefully lower the cathode / top of the semi-dry apparatus on to the gel sandwich.

• Connect the semi-dry blotter to the DC power supply and set transfer conditions between 1.5 – 3.5 mA/cm2 of gel area. Example: A gel

measuring 7cm x 7cm has an area of 49cm2 and will require between 17 and 73mA constant current.

Power conditions and transfer times will vary with DNA/RNA type and size, thickness of the gel and size of the electroblot sandwich. Typically

20 – 60 minutes will be required at current densities of 1.5 – 3.5mA/cm2

During the electrotransfer the voltage should be monitored for large uctuations. During the transfer the voltage will slowly increase to

maintain a constant current. If the voltage is lower than 15V the transfer time will lengthen. If the voltage increases signicantly, such as

greater than 25V, the buer capacity has probably been depleted and the run should be terminated. If the run is not stopped, the gel will

over heat and eventually melt. If the voltage indicated is signicantly lower than normal, for instance less than 15V, the buer solution may be

more concentrated than 1X and therefore less volume may be required to maintain the specic current setting. If this is the case, the run can

be completed as long as the current is adjusted to the specic setting. CARE MUST BE TAKEN NOT TO OVER HEAT THE GEL OR THE BLOTTING

DEVICE. Monitor the procedure closely.

Operational Procedures and

Techniques for Semi-Dry RNA Blotting

Denaturing Formaldehyde Agarose gels

• RNA is separated on a 1.2% Agarose gel with 1X MOPS and 1.8% formaldehyde. (IB70042 – Molecular Biology Grade Agarose &

IB70175 – 10X MOPS w/DEPC water.)

• The Total RNA sample is heated to 65C for 10 – 15 minutes and added to RNA loading buer. Loading buer consists of 50% forma-

mide, 6.5% formaldehyde and 1X MOPS. Typically 5 – 10mg of RNA are loaded per/lane. The samples are then heated to 55C for 10

– 15 minutes and mixed with 1/10 bromophenol blue.

• The gels are electrophoresed in a submarine apparatus in 1X MOPS buer for 3 – 4 hours at 4 – 5v/cm2

• Following electrophoresis, the gel is soaked in 5 gel volumes of 1X TBE buer containing 0.1ug/ml EtBr for 30 minutes. Then soak the

gel in 1X TBE buer without EtBr for another 30 minutes.

• The gel can remain in the 1X TBE until just prior to semi-dry blotting.

• Trim away non-essential portions of the gel. Prepare 2 buer pads, 2 spacer pads and one charge modied nylon membrane cut to

the exact size of the trimmed gel.

• Saturate the trimmed buer pads, two spacer pads and nylon membrane in 1X TBE buer.

• Place one blotter pad on the semi dry blotter anode (base) plate. Roll out any air bubbles that may be trapped beneath the pad. Place

a spacer/thick blot paper on top of the pad. Make certain there are no air bubbles beneath the blotting paper.

• Be certain the membrane has been in TBE buer for 10 minutes to insure equilibration. Place the membrane on top of the blotting

paper making certain there are no air bubbles beneath the membrane.

• Carefully place the equilibrated gel on top of the membrane making certain there are no air bubbles beneath the gel.

• Place a second blot paper/spacer on top of the gel. Place the second blotter pad on top of the blotting paper. Make certain there are

no air bubbles.

• Carefully lower the semi-dry blotter cathode (black top assembly) on to the gel stack.

• Connect the semi-dry apparatus to the power supply and set transfer conditions to 2 – 3.5mA/cm2typical transfer time should 20 – 35

minutes.

Non-denaturing Formaldehyde Agarose gels

• Prior to electrophoresis, heat samples in RNA sample/denaturing buer (4.5ml RNA in ultrapure water (IB42200 – DEPC water –

125ml), 2ml 5X TBE buer 3.5ml formaldehyde and 10ml formamide).

• RNA is separated on a 1 – 1.5% agarose gel, omitting formaldehyde in gel.

• The gel is electrophoresed in a submarine apparatus in 1X TBE buer.

• Following electrophoresis the gel is soaked in 5 gel volumes of 1X TBE buer containing 0.1mg Ethidium Bromide for 30 minutes and

then soaked in 1X TBE buer without Etidium Bromide for 30 minutes.

• The gel should remain in the 1X TBE buer prior to semi-dry blotting.

• Follow the remaining steps above for RNA blotting.

• 1X MOPS buer can be substituted for 1X TBE buer.

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