Innovative and comprehensive analysis kit for Protein S-Palmitoylation ^RapidSPALM, Protein S-Palmitoylation Detection Kit

• What is protein S-palmitoylation?
• Overview of conventional analysis for protein S-palmitoylation
• Overview of ^RapidSPALM
• The principle of ^RapidSPALM Reaction kit
• Feature
• Applications
• Kit selection guide
• Kit information
• Price

What is protein S-palmitoylation?

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Overview of conventional analysis for protein S-palmitoylation

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Overview of ^RapidSPALM

BioDynamics Laboratory Inc., developed a novel chemical strategy called Rapid Substitution of Protein S-Acylation for Multifunctional-tag (^RapidSPALM [rˈæpɪzpάːm]) with only 2-3 hours time-course and highly improved specificity. ^RapidSPALM can converts the S-palmitoyl group on proteins to the unique MfTag which can semi-quantitate total amount of S-palmitoylation, purify comprehensively S-palmitoylated proteins, and count number of S-palmitoyl group. ^RapidSPALM, Protein S-palmitoylation Detection Kit consists of two kit parts, including a Reaction Kit (Cat. No. #F017A) and Purification Kit (Cat. No. #F017B). Firstly, S-palmitoyl groups on proteins in any samples are converted to MfTag by the Reaction Kit. ^RapidSPALM’s MfTag contains three functional units, including a yellow fluorophore (yFL), affinity tag (Hook), and a high molecular weight (~5 kDa) backbone and will label to the cysteine residues, which are originally S-palmitoylated via a disulfide bond. The Hook group on the MfTag can be specifically and quickly isolated by a Loop affinity column provided in Purification Kit. After purification, the MfTag is easily cleaved by reducing reagent.

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The principle of ^RapidSPALM Reaction kit

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, including Acyl-Biotin Exchange (ABE), Acyl-Resin Assisted Capture (Acyl-RAC) and Acyl-PEGyl Exchange Gel Shift assay (APEGS). 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. Owing to the ^RapidSPALM reagents and protocol, ultra-quick and easy preparation is accomplished.

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). Then, hpHA(-) sample (Tris-treatment) is also prepared as a negative control for the estimation of non-specific MfTag labeling.

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. If blocking of non-palmitoyl free-cysteines in Step-3 is insufficient, MfTag non-specifically binds to proteins in the hpHA(-) sample in S-palmitoylation independent manner. MfTag (-) sample is also prepared as a completely negative control.

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

According to reaction procedures above, three control experiments for each sample are required. Hereafter, each control experiment called hpHA(-)/MfTag(-), hpHA(-)/MfTag(+) and hpHA(+)/MfTag(+), and abbreviated as -/-, -/+, and +/+ in the article.

The principle of ^RapidSPALM Purification kit

Hook&Loop affinity purification is based on a non-proteinous and small molecular interaction system. Due to a non-protein-based method, all procedures can be performed under completely denatured conditions, and non-specific binding of applied proteins to affinity beads are highly suppressed. The spin-column format provides an easy procedure, including only the addition of reagent solutions to the column and spinning-down.

Time course comparison between ^RapidS PALM and conventional ABE method

ABE, the most popular conventional detection method for protein S-palmitoylation, requires over one-day procedures from sample preparation to affinity purification. Our ^RapidSPALM kit has ultra-quick procedures and requires only two hours for the MfTag-labeling reaction and an hour for the purification step. Our ^RapidSPALM saves time and labor and provides half-day experiments.

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Feature

• Easy procedure offers only two hours for Reaction Kit and one hour for Purification Kit.＊
• 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.

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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

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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 can perform fluorometric assay and gel shift assay. 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

Purpose	Detection method	Required kit
To compare total amount of S-palmitoylation modification between multiple samples	Fluorometric assay (Measure fluorescent intensity Ex 325/Em 525) by fluorescent spectrometry)	Reaction kit
To detect S-palmitoylated proteins in SDS-PAGE gel	Fluorescent detection in SDS-PAGE gel (Non-reducing SDS-PAGE/Fluorescent imager)	Reaction kit
To estimate number of S-palmitoyl group on target protein	Gel shift assay (Non-reducing SDS-PAGE/WB)	Reaction kit
S-To comprehensively purify S-palmitoylated proteins	Affinity purification and Reducing SDS-PAGE/Silver staining	Reaction kit + Purification kit
To identify S-palmitoylated proteins	Affinity purification and Reducing SDS-PAGE/WB	Reaction kit + Purification kit
To estimate S-palmitoylation level of target protein	Affinity purification and both Reducing and Non-reducing SDS-PAGE/WB	Reaction kit + Purification kit

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Kit information

Kit	Catalog No.	Product name	SizeNote
Reaction Kit	F017A	RapidSPALM, Protein S-Palmitoylation Detection Kit	12 assays
Purification Kit	F017B	RapidSPALM, Additional Components for Affinity Purification	24 columns

Note 1 “1 assay” of Reaction Kit contains reagents enough to prepare three control experiments (-/-, -/+, and +/+) mentioned above from one biological sample.

Note 2 For purification application, “1 assay” basically needs two control experiments (-/+ and +/+) from one biological sample. So, 24 columns (=1 kit) are enough to perform 12 assays.

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Price

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