Invitrogen™ Bolt™ Welcome Pack + iBlot™ 2 System

Catalog No.	For Use With (Equipment)
NW0412AIB2	Mini Gel Tank

Catalog No. NW0412AIB2 Supplier Invitrogen™ Supplier No. NW0412AIB2

Description

A complete protein separation-to-western blot solution by combining a Bolt Welcome Pack A with the iBlot 2 Gel Transfer Device and iBlot 2 Transfer Stack (nitrocellulose, regular size) in lieu of one box of Bolt gels

The Bolt Welcome Pack + iBlot 2 System offers a complete protein separation-to-western blot solution by combining a Bolt Welcome Pack A with the iBlot 2 Gel Transfer Device (Cat. No. IB21001) and iBlot 2 Transfer Stack (nitrocellulose, regular size) (Cat. No. IB23001) in lieu of one box of Bolt gels.

The Bolt Welcome Pack A consists of a Mini Gel Tank plus all of the necessary Bolt™ gels and buffers you will need to get started running gels.

Contents of the Bolt Welcome Pack A:
• Mini Gel Tank (Cat. No. A25977)
• Bolt 4-12% Bis-Tris Plus gels, 10-well, 1 box (Cat. No. NW04120BOX)
• Bolt MES Running Buffer, 20X (Cat. No. B0002)
• Bolt LDS Sample Buffer, 4X (Cat. No. B0007)
• Bolt Sample Reducing Agent, 10X (Cat. No. B0009)
• SeeBlue Plus2 Pre-stained Protein Standard (Cat. No. LC5925)

About the Mini Gel Tank
The Mini Gel Tank is compatible with a variety of Novex gels, NuPAGE gels, and Bolt Bis-Tris Plus gels. Each Mini Gel Tank can accommodate up to two gels per run. The unique tank design enables convenient side-by-side gel loading and enhanced viewing during use. Optimized conditions using constant voltage allow for Bolt Bis-Tris Plus gels to be run in approximately 35 minutes. Run times may vary depending on buffer conditions and power supplies being used.

About Bolt Bis-Tris Plus Gels
Bolt Bis-Tris Plus gels are precast polyacrylamide gels designed for optimal separation of your small- to medium-sized proteins under denaturing conditions. Similar to NuPAGE Bis-Tris gels, Bolt Bis-Tris Plus gels are designed to deliver consistent gel performance and provide a neutral pH environment that minimizes protein modifications. Bolt gels are ideal for western blot transfer and analysis, and any other techniques where protein integrity is crucial. Also use Bolt gels to obtain optimal results for your day-to-day protein separation needs. Bolt Bis-Tris Plus gels come in four g types and multiple well formats. Each box contains 10 gels.

About the iBlot 2 Gel Transfer Device
The iBlot 2 Gel Transfer Device is our second-generation dry-transfer device, providing the same speed and convenience as the original iBlot device, but with many new features. The iBlot 2 Gel Transfer Device performs western blotting transfer simply, efficiently, and reliably, within seven minutes or less, and without the need for liquid buffers. The iBlot 2 Gel Transfer Device is an integral part of the iBlot 2 Dry Blotting System, which consists of the transfer device and consumable transfer stacks that contain the required buffers and transfer membrane (nitrocellulose or PVDF).

Specifications

• For Use With (Equipment): Mini Gel Tank
• Gel Size: Mini
• Membrane Compatibility: Nitrocellulose
• Product Line: Bolt, iBlot2
• Gel Percentage: 4 to 12%
• Gel Type: Bis-Tris Plus
• No. of Wells: 10-well
• Quantity: 1 Welcome Pack
• Separation Range: 3.5 to 260 kd
• Shipping Condition: Approved for shipment at room temperature or on wet ice
• Thickness (Metric): 1.0 mm

Frequently Asked Questions (FAQs)

If a green color appears on the membrane after the transfer using the iBlot 2 Dry Blotting System, will it affect the results?

The green discoloration is copper deposits from the transfer stack, and it does not affect the results. To minimize this effect, shake excess water off the filter paper and buffer from the gel before placing each on the stack. The current formulation of stacks minimizes the green discoloration.

Can I cut the bottom stack of my iBlot 2 Transfer Stack to fit my separating gel?

No. Do not trim the iBlot 2 Transfer Stacks to fit your gel size.

Is the PVDF membrane in the iBlot 2 PVDF Gel Transfer Stack pre-activated?

The PVDF membrane is pre-activated and ready for use without any pre-treatment with alcohol.

Can I use the stack and/or absorbent pad more than once when using the iBlot 2 Dry Blotting System?

No. The transfer stacks have a finite amount of ions to drive the transfer and are depleted after a single use.

Can I perform multiple transfers, with a new transfer starting immediately after the previous one when using the iBlot 2 Dry Blotting System?

Yes. The device is designed to do so with no impact on performance.

Can I create my own transfer conditions with the iBlot 2 Dry Blotting System?

Yes. The device allows programming custom methods.

Can I prepare my protein sample with the reducing agent and store it for future use?

DTT is not stable, so it must be added and the reduction performed just prior to loading your samples.

My LDS or SDS sample buffer precipitates when stored at 4 degrees C. Can I warm it up? Can I store it at room temperature?

Precipitation of the LDS or SDS at 4 degrees C is normal. Bring the buffer to room temperature and mix until the LDS/SDS goes into solution. If you do not want to wait for it to dissolve, you can store the sample buffer at room temperature.

What are the storage conditions for Bolt gels?

Similar to NuPAGE gels with storage temperatures of 4 to 25 degrees C.

I am transferring a Tris-Glycine gel using constant voltage and the current reading is way over the expected starting current. Can you offer some suggestions?

The most common cause of abnormally high current is the transfer buffer. If the transfer buffer is too concentrated, this leads to increased conductivity and current. High current may also occur if Tris-HCl is accidentally substituted for the Tris base required in the transfer buffer. This will again result in low buffer pH and lead to increased conductivity and current and subsequently, overheating. We recommend checking the transfer buffer and its reagent components and re-diluting or remaking the buffer.

I ran my protein under native conditions on a Tris-Glycine gel. It has a pI that is higher than the pH of the Tris-Glycine transfer buffer. Can you offer some tips for transferring it?

- Increase the pH of Tris-Glycine transfer buffer to 9.2, allowing all the proteins below pI 9.2 to transfer towards the anode electrode.
- Use the Tris-Glycine transfer buffer and place a membrane on both sides of the gel. If there are any proteins that are more basic than the pH of the transfer buffer, they will be captured on the extra membrane placed on the cathode side of the gel. Both membranes can then be developed in the same manner.
- Prior to blotting, incubate the gel for 15 minutes in Tris-Glycine transfer buffer containing 0.1% SDS. The small amount of SDS will give the proteins enough charge to move unidirectionally towards the anode and in most cases, should not denature the protein. Proceed with the transfer using regular Tris-Glycine transfer buffer.

I had problems transferring my larger-molecular weight proteins from my NuPAGE gel. Can you please offer some suggestions?

For proteins larger than 100 kDa, we recommend pre-equilibrating the gel in 2X NuPAGE Transfer buffer (without methanol) containing 0.02-0.04% SDS for 10 minutes before assembling the sandwich and then transferring using 1X NuPAGE transfer buffer containing methanol and 0.01% SDS.

What causes empty spots on my membrane after transfer?

Here are possible causes and solutions:

- Presence of air bubbles between the gel and the membrane preventing the transfer of proteins. Be sure to remove all air bubbles between the gel and membrane by rolling a glass pipette over the membrane surface.
- Expired or creased membranes used. Use fresh, undamaged membranes.

I performed a western transfer and see the appearance of diffuse bands and swirling patterns on the membrane. What could have happened?

The swirling and diffuse banding patterns are typical of molecules moving laterally before binding to the membrane during transfer. Here are possible causes and solutions:

- Poor contact between the gel and the membrane: The gel should be attached to the membrane through capillary action. To ensure that this happens, make sure that you roll over the surface of each layer of the gel/membrane sandwich with a glass pipette to ensure good contact between the gel and the membrane. It is helpful to use a disposable pipette to place some extra transfer buffer on the surface of each layer as the sandwich is being made. Also, the pads need to be fully saturated (push down with gloved hand when they are placed in transfer buffer to make sure there are no air bubbles.)
- Under-compression of the gel: The gel/membrane assembly should be held securely between the two halves of the blot module. Try adding another pad or replace any pads that have lost their resiliency with fresh ones.
- Over-compression of the gel: A good indication of over-compression is if the gel has been excessively flattened. In the event that the sandwich is over-compressed, remove enough pads so that the blotter can be closed without exerting excess pressure on the gel and membrane.
Note: The height of the uncompressed pads should be 0.5-1.0 cm above the level of the sealing gasket.

When I perform a western transfer, the power supply shuts off in the middle of the transfer. What is wrong?

Here are possible causes and solutions:

- High ionic strength of the transfer buffer. Prepare the buffer as described in the manual.
- Power supply is operating at a current close to the current limit of the power supply. Use a power supply with higher limits.

After a western transfer, I noticed that a significant amount of protein remained in the gel indicated by staining of the gel after transfer. What should I do?

Here are possible causes and solutions:

- Too short a transfer time: Increase the blotting time by 15 minute increments.
- Inappropriate gel type: Check the percentage of the gel used and switch to a higher percentage gel.
- Inappropriate amount of SDS: Add 0.01-0.02% SDS to the transfer buffer to facilitate migration of the protein out of the gel.
- Inappropriate methanol content: Decrease the amount of methanol in the transfer buffer.
Note: Higher molecular weight proteins usually do not transfer completely as compared to mid to low molecular weight proteins.

After a western transfer, I noticed that a significant amount of protein had passed through the membrane indicated by the presence of proteins on the second membrane. Can you please help?

Here are possible causes and solutions:

- Too long a transfer tim: Shorten the transfer time by 15 minute increments.
- Inappropriate amount of SDS: Do not include any SDS in the transfer buffer.
- Inappropriate methanol content: Add additional methanol to the transfer buffer to increase the binding capacity of the membrane.
- Inappropriate gel type: Check the percentage of the gel used and switch to a higher percentage gel.
- Sample overloaded: Decrease the sample load.
- Finally, if using nitrocellulose membrane, switch to PVDF which has a higher binding capacity.

I performed a western transfer and none of my proteins transferred to the membrane. Can you offer some tips?

It is possible that the gel/membrane sandwich was assembled in the reverse direction such that the proteins have migrated out into the buffer. Assemble the blot sandwich in the correct order using instructions provided in the manual.

During western transfer conditions using constant voltage, what would cause the actual current to greatly exceed the expected starting current?

The most common cause of abnormally high current is the buffer. If the buffer is too concentrated, this leads to increased conductivity and higher current. High current may also occur if Tris-HCl was accidentally substituted for the Tris base required in the transfer buffer. Tris-HCl results in a low buffer pH and leads to increased conductivity and current, and, subsequently, overheating. Check the transfer buffer and its reagent components, re-dilute, or remake the buffer.

I am transferring my gel using constant voltage and the current reading has dropped much lower than the expected starting current. What could have happened?

Here are possible causes and solutions:

- The buffer was accidentally made too dilute, therefore increasing resistance and thus lowering conductivity and current: Check the transfer buffer and its reagent components and then re-dilute it or remake it.
- The circuit is broken or impeded, as in the case of a corroded or broken electrode or malfunctioning power supply: Check the equipment.
- There is a leak in the blot module (this is indicated by a drastic decrease in current and in buffer volume within the module): Ensure that the inner buffer chamber is filled sufficiently so that the wells are covered with buffer.
- Tape at the bottom of the gel cassette was not removed: Double check that the tape on the bottom of the gel has been removed.

For Research Use Only. Not for use in diagnostic procedures.