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S-Palmitoylation

Analysis / Purification kit for Protein S-Palmitoylation

RapidSPALM, Protein S-Palmitoylation Detection Kit

Highlights

  • Higher specificity and higher protein recovery compared to conventional methods

  • Only two hours for Reaction Kit and one hour for Purification Kit. (estimate for one assay)

  • Comprehensive applications can be performed by only one platform.

  • A wide range of samples, including animal tissue, cultured cells, plant tissues, etc., can be applied.

  • As spin-column format in the purification kit provide easy and quick procedures.

Product Information

RapidS PALM, Protein S-Palmitoylation Detection Kit is an innovative analysis kit for the protein S-palmitoylation, including S-acylation. RapidSPALM kit converts the S-palmitoyl/acyl group on proteins to a unique multifunctional-tag (hereafter MfTag) and provides information about total S-palmitoylation amounts in multiple samples, comprehensive S-palmitoylated proteins, number of S-palmitoyl group on target proteins, and ratio of S-palm/non-palm form.

Compared to conventional methods such as Acyl-Biotin Exchange (ABE), Acyl-Resin Assisted Capture (Acyl-RAC) and Acyl-PEGyl Exchange Gel Shift assay (APEGS), RapidSPALM show following advantages.
  1. Higher tag labeling efficiency

  2. Lower non-specific modification

  3. Significantly shorter reaction times and steps

  4. Simple and Fast preparation with column

  5. Higher recovery in the purification steps

<The principle of RapidSPALM>

Our RapidSPALM Reaction kit converts S-palmitoyl groups on proteins to a unique MfTag by stepwise chemical reactions similar to conventional S-palmitoylation detection methods. Unlike ABE, Acyl-RAC or APEGS, RapidSPALM includes several original and high-performance reagents with a highly optimized protocol and will reduce non-specific labeling reaction, dramatically shorten each reaction time, and omit time-consuming steps.

・Reaction kit

Step-1 Strong denaturation of proteins
A strong denaturant and heat denature proteins to increase the accessibility of the following reagents.

Step-2 Reduction of disulfide bond
Disulfide bonds are cleaved by a potent reducing reagent.

Step-3 Blocking of free cysteines
Unique and high-performance thiol-specific reagents block free cysteines.

Step-4 Cleavage of S-palmitoyl thioester
S-palmitoyl thioesters are cleaved by our high performance hydroxylamine-derivative (hpHA).

Step-5 MfTag labeling
Free-cysteines newly generated from the hpHA cleavage of S-palmitoyl thioester are labeled with MfTag by a cysteine-specific MfTag-labeling reagent.

Step-6 Desalting unreacted reagents by Chloroform/Methanol precipitation (CMppt)

・Additional purification kit

Step-7 Affinity purification
Hook&Loop affinity purification is based on a non-proteinous and small molecular interaction system. Non-specific binding of applied proteins to affinity beads are highly suppressed and the spin-column format provides an easy procedure.

Step-8 Removal of MfTag
MfTag can be removed by reducing agent.

<Kit selection guide>

There are two parts of RapidSPALM kit, Reaction kit and Purification kit. Reaction kit is an essential part to perform RapidSPALM experiment and Purification kit is an optional component to perform affinity purification. Please select adequate kit format according to experimental purposes listed in the Table below.

In RapidSPALM experiment, reducing or non-reducing condition on SDS-PAGE is important depending on experimental purposes. Please confirm the recommended condition on your experiments.

Note: Purification Kit alone could not prepare RapidSPALM experiments. You need Reaction Kit alone or pair of Reaction Kit and Purification Kit.

Table Selection guide by experimental purpose

<Applications>

・Quantification of total S-palmitoylation amounts in samples by fluorometric measurement
・Detection of S-palmitoylation protein bands in SDS-PAGE gel by fluorometric imager
・Estimation of S-palmitoyl group number on target proteins by gel shift assay
・Comprehensive purification of S-palmitoylated proteins by affinity column
・Estimation of S-palmitoylated ratio of target proteins by affinity column and western blotting


Code

Size

12 assays

Manuals

SDS