SBP | Sedoheptulose-1,7-bis phosphatase positive control/quantitation standard

AS15 2873S | protein standard for quantitation and positive control

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product information
Background		SBPase (Sedoheptulose-1,7-bis phosphatase) is a chloroplast enzyme involved in the carbon reduction of the Calvin cycle, part of carbohydrate metabolism. Source of SBPase standard: SBPase standard source is Sphingomonas wittichii strain RW1, overexpressed in E.coli bearing an N-terminal his6 tag.
Host
Clonality
Clone
Purity
Format		Lyophilized
Quantity
Reconstitution		For reconstitution add 85 µl of sterile water. Please note that this product contains glycerol and might appear as liquid but is provided lyophilized.
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		Primary antibodies matching SBPase standard are: AS15 2873 \| anti-SBPase \| Sedoheptulose-1,7-bis phosphatase , rabbit antibodies collection of other protein standards collection of matching global antibodies Plant and algal protein extraction buffer
Additional information		The SBPase calibrated protein standard can be used in combination with Agrisera global anti-SBPase antibiodies (AS15 2873) to quantitate SBPase from a wide range of species. Global antibodies are raised against highly conserved amino acid sequence. 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/well will give a RbcL signal in this range. Positive control: a 2 μl load per well is optimal for most chemiluminescent detection systems. Higher standard concentration needs to be used to allow detection by Coomasie stains. Such gels with higher standard concentration can not be used for quantitation using chemiluminescence. 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.
Expected \| apparent MW		27 kDa
Confirmed reactivity
Predicted reactivity
Not reactive in
Additional information		Concentration: after re-constitution with sterile milliQ water final concentration of the standard is 0.15 pmoles/µl 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), 50 mM 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		to be added when available, standard available in November 2015

application example

western blot using anti-SBP antibodies

Following standard western blot procedure this image was obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad) as described below. 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.

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 applied to the area for quantitation and the values are background subtracted to give an adjusted volume in counts for each standard and sample. Using this 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