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Invitrogen™ SureLock Retrofit Kit for XCell II™ Mini-Cell

Catalog No. EI0020
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Catalog No. EI0020 Supplier Invitrogen™ Supplier No. EI0020
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Description

Includes

SureLock™ mini-cell lid, gel tension wedge, and molded mini-gel buffer dam.

Converts an XCell II™ Mini-Cell into an XCell SureLock™ Mini-Cell

1D Gel Electrophoresis, Protein Gel Electrophoresis, Proteins, Expression, Isolation and Analysis

Order Info

Shipping condition: Room Temperature

Specifications

Content And Storage Store at room temperature.
For Use With (Application) 1D Gel Electrophoresis, Protein Gel Electrophoresis, Proteins, Expression, Isolation and Analysis
For Use With (Equipment) XCell SureLock™ Mini
Gel Size Mini
Product Line XCell II, XCell SureLock
Type Xcell Retrofit Kit
Quantity 1 unit
Shipping Condition Room Temperature
Max. Voltage 1500 V

Frequently Asked Questions (FAQs)

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.
Can I purchase replacement parts for the XCell SureLock Mini-Cell?

Here are the replacement parts we offer for the XCell SureLock Mini-Cell:
Replacement part - Cat. No.
Buffer Dam for XCell SureLock Mini-Cell and XCell II Blot Module - EI0012
L-Buffer Chamber for XCell SureLock Mini-Cell and XCell II Mini-Cell - EI0013
Platinum Wire for XCell SureLock Mini-Cell (12 inch x 2) - EI9022
XCell SureLock Lid - EI0010
XCell SureLock Wedge - EI0011
Buffer Core with Cathode Bar for XCell SureLock Mini-Cell - EI9015
Gel Knife - EI9010
Sponge Pad for Blotting (8) - EI9052
How do I convert my old XCell II Mini-Cell to an XCell SureLock Mini-Cell?

The XCell II Mini-Cell has been discontinued and can be upgraded to an XCell SureLock Mini-Cell with the purchase of the SureLock Retrofit kit for XCell II Mini-Cell, Cat. No. EI0020 (consists of a SureLock mini-cell lid, gel tension wedge, and buffer dam).
What are the components of the XCell SureLock Mini-Cell?

The XCell SureLock Mini-Cell includes the lid, gel tension wedge, lower buffer chamber, buffer core, buffer dam, and gel knife.

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