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PsaC | positive control/quantitation standard

238 €

AS04 042S  |  recombinant protein standard for quantitation


PRODUCT INFORMATION IN PDF

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Item No:
AS04 042S

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product information
Background
PsaC is a conserved, chloroplast-encoded, Fe-S binding protein of approximately 10kDa, present in all known Photosystem I complexes. It is located on the stromal side of the thylacoid membranes. PsaC coordinates the Fe–S clusters FA and FB through two cysteine-rich domains.

This product is a recombinant protein standard, source: Synechocystis PCC 6803.
Host
Clonality
Clone
Purity
Format
Quantity 100 µl
Reconstitution
For reconstitution add 95 µl of milliQ water. Please note that this product contains 10% glycerol and might appear as liquid but is provided lyophilized. PsaC standard protein concentration: 0.10 pmol/µl. 
Storage

Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.

Tested applications
Western Blot (WB)
Related products

AS04 042
| anti-PsaC | PSI-C core subunit of photosystem I global rabbit antibodies
Additional information

The PsaC protein standard can be used in combination with global anti-PsaC antibodies to quantitate PsaC from a wide range of species. Global antibodies are raised against highly conserved amino acid sequences in the PsaC protein.

Quantitative western blot:  detailed method description, video tutorial

application information
Recommended dilution
Standard curve: 3 loads are recommended (0.5, 2 and 4μl).

For most applications a sample load of 0.2 μg of chlorophyll will give a PsaC signal in this range.

Positive control: a 2 μl load per well is optimal for most chemiluminescent detection systems.

This standard is ready-to-load and does not require any additions or heating.

Please note that this product contains 10% glycerol and might appear as liquid but is provided lyophilized. Allow the product several minutes to solubilize after adding water. Mix thoroughly but gently Take extra care to mix thoroughly before each use, as the proteins tend to settle with the more dense layer after freezing.
Expected | apparent MW

11.5 kDa (larger than native protein due to the addition of His-tag). In most gels PsaC migrates between 9 and 14 kDa

Confirmed reactivity
Predicted reactivity
Not reactive in
Additional information

Protein standard buffer composition: Protein standard buffer composition: Glycerol 10%, Tris Base 141 mM, Tris HCl 106 mM, LDS 2%, EDTA 0.51 mM, SERVA® Blue G250 0.22 mM, Phenol Red 0.175 mM, pH 8.5, 0.1mg/ml PefaBloc protease inhibitor (Roche), 50mM DTT.

This standard is ready-to-load and does not require any additions or heating. It needs to be fully thawed and thoroughly mixed prior to using. Avoid vigorous vortexing, as buffers contain detergent. Following mixing, briefly pulse in a microcentrifuge to collect material from cap.

This standard is stabilized and ready and does not require heating before loading on the gel.

Please note that this product contains 10% glycerol and might appear as liquid but is provided lyophilized. Allow the product several minutes to solubilize after adding water. Mix thoroughly but gently Take extra care to mix thoroughly before each use, as the proteins tend to settle with the more dense layer after freezing.

Selected references Li et al. (2016). A Hard Day's Night: Diatoms Continue Recycling Photosystem II in the Dark. Front. Mar. Sci., 08 November 2016 | http://dx.doi.org/10.3389/fmars.2016.00218
Vandenhecke et al. (2015). Changes in the Rubisco to photosystem ratio dominates photoacclimation across phytoplankton taxa. Photosynth Res. 2015 Apr 11.
Wu et al. (2014). Large centric diatoms allocate more cellular nitrogen to photosynthesis to counter slower RUBISCO turnover rates. Front. Mar. Sci., 09 December 2014 | doi: 10.3389/fmars.2014.00068.
Li et al. (2014). The nitrogen costs of photosynthesis in a diatom under current and future pCO2. New Phytol. 2014 Sep 25. doi: 10.1111/nph.13037.

Application example

total protein from Trichodesmium sp. (1) and Thalassiosira sp (2). Recombinant PsaC protein standard (AS04 042S) (3-6) loaded at 0.5 pmoles, 0.3 0.1 and 0.05 pmoles. Molecular weight markers (MagicMark XP, Invitrogen) (7).  Samples were separated on  4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in 2% ECL Advance blocking reagent (GE Healthcare) in 20 mM Tris, 137 mM sodium chloride pH 7.6 with 0.1% (v/v) Tween-20 (TBS-T) for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 50 000 for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (anti-hen IgY horse radish peroxidase conjugated, from Abcam) diluted to 1:50 000 in 2% ECL Advance blocking solution for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with ECL Advance detection reagent according the manufacturers instructions. Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad).

Note: Optimal quantitation is achieved using moderate sample loads per gel lane, generally 0.5 to 2.5 ug total protein, depending on the abundance of the target protein.

 

  western blot detection with PsaC protein standard

 

Quantitation: When quantitated standards are included on the blot, the samples can be quantitated using the available software. Excellent quantitation can be obtained with images captured on the Bio-Rad Fluor-S-Max or equivalent instrument using Bio-Rad QuantityOne software. The contour tool is used to select the area for quantitation and the values are background subtracted to give an adjusted volume in counts for each standard and sample. Using above protocol linear standard curves are generated over 1-1.5 orders of magnitude range in target load. It is important to note that immunodetections usually show a strongly sigmoidal signal to load response curve, with a region of trace detection of low loads, a pseudolinear range and a region of saturated response with high loads. For immunoquantitation it is critical that the target proteins in the samples and the standard curve fall within the pseudolinear range. Our total detection range using this protocol spans over 2 orders of magnitude, but the quantifiable range is narrower.

Quantitative western blot:  detailed method description.


||| For other applications, usage on species other than stated above or any other questions, please use the LiveChat option or contact us at support@agrisera.com