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50ug
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KCNN3 antibody - C-terminal region (ARP35097_P050)

Description of Target:
Action potentials in vertebrate neurons are followed by an afterhyperpolarization (AHP) that may persist for several seconds and may have profound consequences for the firing pattern of the neuron. Each component of the AHP is kinetically distinct and is mediated by different calcium-activated potassium channels. KCNN3, a member of the KCNN family of potassium channel genes, encodes a protein that is activated before membrane hyperpolarization and is thought to regulate neuronal excitability by contributing to the slow component of synaptic AHP. The encoded protein is an integral membrane protein that forms a voltage-independent calcium-activated channel with three other calmodulin-binding subunits.
Gene Symbol:
KCNN3
Official Gene Full Name:
Potassium intermediate/small conductance calcium-activated channel, subfamily N, member 3
NCBI Gene Id:
3782
Alias Symbols:
SK3; hSK3; SKCA3; KCa2.3
Sample Type Confirmation:

KCNN3 is supported by BioGPS gene expression data to be expressed in Daudi

Tissue Tool:
Find tissues and cell lines supported to express KCNN3.
Protein Accession #:
NP_002240
Nucleotide Accession#:
NM_002249
Swissprot Id:
Q9UGI6
Host:
Rabbit
Clonality:
Polyclonal
Protein Name:
Small conductance calcium-activated potassium channel protein 3
Protein Size (# AA):
736
Molecular Weight:
81kDa
Application:
IHC, WB
Partner Proteins:
CALM1, PRKAG1, UBC
Immunogen:
The immunogen for anti-KCNN3 antibody: synthetic peptide directed towards the C terminal of human KCNN3
Product Format:
Lyophilized powder
Purification:
Affinity Purified
Complete computational species homology data:
KCNN3 antibody - C-terminal region (ARP35097_P050)
Predicted Homology Based on Immunogen Sequence:
Dog: 100%; Pig: 100%; Rat: 100%; Human: 100%; Mouse: 100%; Bovine: 100%; Rabbit: 100%; Guinea pig: 100%; Horse: 93%
Species Reactivity:
Bovine, Dog, Pig, Rabbit, Rat, Guinea pig, Mouse, Human, Horse
Datasheets / Downloads:
Printable datasheet for
anti-KCNN3 antibody
- ARP35097_P050
Peptide Sequence:
Synthetic peptide located within the following region: ITELNDRSEDLEKQIGSLESKLEHLTASFNSLPLLIADTLRQQQQQLLSA
Blocking Peptide:
For anti-KCNN3 antibody is Catalog # AAP35097 (Previous Catalog # AAPP06328)
Target Reference:
Kolski-Andreaco,A., et al., (2004) J. Biol. Chem. 279 (8), 6893-6904
Reconstitution and Storage:
Add 50 ul of distilled water. Final anti-KCNN3 antibody concentration is 1 mg/ml in PBS buffer with 2% sucrose. For longer periods of storage, store at -20C. Avoid repeat freeze-thaw cycles.

Product Review: KCNN3 antibody - C-terminal region (ARP35097_P050) tested with mouse brain slices in Western blot

Product Page Link: KCNN3 antibody - C-terminal region (ARP35097_P050)

Data Provided by Eric Lloyd of Baylor College of Medicine

What type of exerimental sample are you using and how did you prepare it?

My preparation involved isolating RNA from mouse kidney, aorta and brain from KCNK6 wildtype and knockout mice.
RNA from whole brain and whole kidney were isolated using Trizol reagent according to the manufacturers instructions. RNA from aorta was oatained using Qiagen’s Micro RNA isolation kit according to the manufacturers’ instructions.

What applications did you test the antibody in? Please include dilutions of the primary and secondary reagents.

I tested the antibodies for Western blot analysis. I used the Aviva-recommended dilutions as follows:

ARP35097_P050 (2 ug/ml)

The Pierce Super Signal West Fempto kit was used. The secondary antibody was goat anti-rabbit and used at 1:10,000 dilution

What controls were used in your experiment? Please include your positive control.

GAPDH was used as the loading control. Tissues known to express the targets were used as the positive control and tissues known to not express the targets were used as the negative control.

ARP35097_P050 –anti KCNN3 (antibody 1). This antibody was tested on wildtype mice as a knockout is not available in

How did you store the antibody after re-suspension?
The antibody was stored at 4C after resuspension and was used the same day it was reconstituted and the day after.

Please provide the protocol for your application procedure. Please be as detailed as possible.

Prepare Tbuss buffer (for protein extraction)
20mM Tris pH 8.0 or 10mM NaH2PO4 pH 7.8
150 mM NaCl
1 mM DTT
1% NaDeoxycholate
0.5% SDS
1% NP40
Add
Aprotinin 10 mg/ml stock
Use 1:5000
Leupeptin 5 mg/ml stock
Use 1:1000
PMSF 100mM stock
Use 1:200
Prepare gel loading buffer
5x protein loading dye (for 10 mls)
250 mM Tris pH 6.8 (2.5 mls 1 M tris pH 6.8)
500 mM DTT (0.77 g)
10% SDS (1g)
0.5% Bromophenol blue (50mg)
50% glycerol (5 mls)
Water (2.5 mls)

Protein extraction protocol
1. Protein extraction from Tissues
i. Add 10X volume tissue with Tbuss and inhibitors (use Roche Complete mini EDTA-free made fresh in TBuss)
1. Use Dounce homogenizer to gently run 5-strokes to lyse cells
ii. Transfer to screw cap epi
iii. Boil 10min
iv. Spin 10 min 4C
v. transfer supernanent to new tube
vi. Take 20ul aliquot for [protein] determination
vii. Freeze at –70C until ready to perform WB
viii. Freeze-thaw only once

2. Lowry assay
1. Biorad Dc protein determination assay
i. Add 20 ul S to each ml reagent A
ii. Add 50 ul reagent A / 10k cells (if 100ul is used per p33 dish, then there is about 10k cells /ul Tbuss
1. Include dilutions of 1:2 and 1:10 etc
iii. Perform standard between 0.2 and 1.5 ug/ul
1. Therefore for total volume of 230ul = from 46ug to 368 ug per well
iv. Begin with 5 ul sample or standard and add 25 ul A
1. Dilute sample 1:0, 1:2, 1:10 (therefore 5 ul, 2.5 + 2.5 water, and 0.5 + 4.5 water) in triplicate
v. Then add 200 ul B and shake 5 sec
vi. Wait 15 min then read A750

96-well format
ug std Air Air Blank 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Air Air Blank 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Air Air Blank 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Air Air Blank 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Sample S1a S1b S1c S2a S2b S2c S3a S3b S3c S4a S4b S4c
a = 1:0 S1a S1b S1c S2a S2b S2c S3a S3b S3c S4a S4b S4c
B = 1:1 S1a S1b S1c S2a S2b S2c S3a S3b S3c S4a S4b S4c
C = 1:10 S1a S1b S1c S2a S2b S2c S3a S3b S3c S4a S4b S4c

3. Western blot

1. Use 6x protein loading buffer (or Laemmle) to sample or standard
i. Aim for 20ul total loading volume therefore add 6 ul loading buffer and 20ul sample- gives enough for error for Westerns
1. Load 5-10 ug/well
ii. Standard = SDS-page broad range marker 6.5-200kDa
1. Dilute 1:20 in Laemmle and heat 95C 5 min
2. Cool and load 5ul/well for minigels and 10ul per well for full length
2. samples
i. Remove protein from –20C and thaw
ii. Heat block to 85C
iii. Add 4ul loading buffer in 20ul sample

1. Alternately may use Laemmle buffer at 1:1 and boil 5 min –then proceeding directly to vii quickly with warm samples that were pulsed down
iv. Heat 85C 10 min
v. Spin pulse 2-3 seconds
vi. Ice samples until gel
vii. Load gel wells
viii. Run 90V ‘till the dye front runs off
ix. Setup transfer apparatus
x. Transfer o/n 20V 4C
xi. Stain with Panceau 5 min
xii. Wash in ddH2O and take copy of blots
xiii. Wash in PBT (0.1% tween)
xiv. Block 1 hour (Pierce Western block or 5% NFDM)
xv. Apply primary antibody in block for 1 hour
xvi. Wash PBT 20min x3
xvii. Apply secondary antibody for 1 hour
xviii. Wash 20 min x’s three

1. Use Pierce femptomol kit
a. First probe for unknown
i. Anti rabbit 1:10K 60 min RT
b. Then strip blot and reprobe for GAPDH
i. GAPDH 1:20K 30 min with HRP direct
2. Mix pierce kit 1:1 and incubate on blot 1 min
3. Image on film and scan image to computer
4. Overlay panceau and mark protein ladder standards
3. Store membranes 4C in wet wrap
4. Stripping PVDF membrane of fluorescence conjugate
i. Use Pierce stripping buffer 30 min RT followed by PBST rinses
1. You do not need to reblock following stripping
2. Continue on to probing step

Please provide an image of your results. How does it compare to your previous images?
The KCNN3 antibody (ARP35097_P050) worked good.

How would you rate this antibody on a scale from 1-5? Why?
I would rate ARP35097 with a 5. There was a clear band at the correct apparent molecular weight. The protein was found in brain but not in kidney and non-specific staining was a minimum.

Would you use this antibody in future experiments?
Yes.

Have you used another antibody which has worked in your application?
I have used several antibodies from various vendors for K channels. There are varying degrees of success. However, an antibody for KCNK6 that is functional does not exist.

Computational species homology for KCNN3 antibody (ARP35097)

Product page for KCNN3 antibody (ARP35097)

The information below lists the predicted species and target name associated to the peptide antigen. Please note, all available target reference numbers to the antigen sequence are presented, including unreviewed and protein isoforms.
To search for antibodies by species, please visit Aviva’s Species Reactivity Page. To search Aviva’s catalog of antibodies by sequence, please visit Aviva’s Antibody Blast Tool.

Predicted Species & Target Target Reference Predicted Homology
African elephant KCNN3 antibody; Loxodonta africana KCNN3 antibody G3TYA7 100%
African elephant KCNN3 antibody; Loxodonta africana KCNN3 antibody G3T5E2 100%
Bovine KCNN3 antibody; Bos taurus KCNN3 antibody E1BHB0 100%
Dog KCNN3 antibody; Canis familiaris KCNN3 antibody E2RQI5 100%
Giant panda LOC100481356 antibody; Ailuropoda melanoleuca LOC100481356 antibody D2HJN4 100%
Gray short-tailed opossum KCNN3 antibody; Monodelphis domestica KCNN3 antibody F6QE12 100%
Guinea pig KCNN3 antibody; Cavia porcellus KCNN3 antibody H0VU03 100%
Horse LOC100057020 antibody; Equus caballus LOC100057020 antibody F7AWG9 92%
Human KCNN3 antibody; Homo sapiens KCNN3 antibody Q9UGI6 100%
Human KCNN3 antibody; Homo sapiens KCNN3 antibody Q9UGI6-2 100%
Human KCNN3 antibody; Homo sapiens KCNN3 antibody Q96DJ3 100%
Human KCNN3 antibody; Homo sapiens KCNN3 antibody Q86VF9 100%
Human KCNN3 antibody; Homo sapiens KCNN3 antibody Q6JXY2 100%
Little brown bat KCNN3 antibody; Myotis lucifugus KCNN3 antibody G1NXP9 100%
Lowland gorilla KCNN3 antibody; Gorilla gorilla gorilla KCNN3 antibody G3S7U0 100%
Lowland gorilla KCNN3 antibody; Gorilla gorilla gorilla KCNN3 antibody G3RAN8 100%
Mouse KCNN3 antibody; Mus musculus KCNN3 antibody P58391 100%
Mouse Kcnn3 antibody; Mus musculus Kcnn3 antibody Q45FA7 100%
Mouse Kcnn3 antibody; Mus musculus Kcnn3 antibody Q3UUY9 100%
Northern white-cheeked gibbon KCNN3 antibody; Nomascus leucogenys KCNN3 antibody G1RL54 100%
Pig KCNN3 antibody; Sus scrofa KCNN3 antibody P58392 100%
Pig KCNN3 antibody; Sus scrofa KCNN3 antibody F1RGN2 100%
Pig KCNN3 antibody; Sus scrofa KCNN3 antibody F1RGN1 100%
Pig KCNN3 antibody; Sus scrofa KCNN3 antibody F1RGN0 100%
Rabbit KCNN3 antibody; Oryctolagus cuniculus KCNN3 antibody G1T5M7 100%
Rabbit LOC100346635 antibody; Oryctolagus cuniculus LOC100346635 antibody G1TJZ5 100%
Rat KCNN3 antibody; Rattus norvegicus KCNN3 antibody P70605 100%
Rat Kcnn3 antibody; Rattus norvegicus Kcnn3 antibody G3V8S7 100%
Rat Kcnn3 antibody; Rattus norvegicus Kcnn3 antibody Q3S913 100%
Rhesus macaque Mmu.3789 antibody; Macaca mulatta Mmu.3789 antibody F7ETY4 100%
Rhesus macaque Mmu.3789 antibody; Macaca mulatta Mmu.3789 antibody F7ETX9 100%
Rhesus macaque SK3 antibody; Macaca mulatta SK3 antibody Q646T9 100%
Small-eared galago KCNN3 antibody; Otolemur garnettii KCNN3 antibody H0WSE3 100%
Tasmanian devil KCNN3 antibody; Sarcophilus harrisii KCNN3 antibody G3WPX5 100%
White-tufted-ear marmoset LOC100387208 antibody; Callithrix jacchus LOC100387208 antibody F7I0V6 100%
White-tufted-ear marmoset LOC100387208 antibody; Callithrix jacchus LOC100387208 antibody F7I0V2 100%
White-tufted-ear marmoset LOC100387208 antibody; Callithrix jacchus LOC100387208 antibody F7E5T3 100%
White-tufted-ear marmoset LOC100387208 antibody; Callithrix jacchus LOC100387208 antibody F6PSC8 100%
Zebra finch KCNN3 antibody; Taeniopygia guttata KCNN3 antibody H0Z127 85%

Product Protocols: KCNN3 antibody tested with Human Daudi Cells (ARP35097_P050)

Aviva Systems Biology is the original manufacturer of this KCNN3 antibody (ARP35097_P050)

Click here to view the KCNN3 antibody Western Blot Protocol

Product Datasheet Link: KCNN3 antibody (ARP35097_P050)

WB Suggested Anti-KCNN3 Antibody Titration: 0.2-1 ug/ml
ELISA Titer: 1:62500
Positive Control: Daudi

Western Blot image:


Description of Target: Action potentials in vertebrate neurons are followed by an afterhyperpolarization (AHP) that may persist for several seconds and may have profound consequences for the firing pattern of the neuron. Each component of the AHP is kinetically distinct and is mediated by different calcium-activated potassium channels. KCNN3, a member of the KCNN family of potassium channel genes, encodes a protein that is activated before membrane hyperpolarization and is thought to regulate neuronal excitability by contributing to the slow component of synaptic AHP. The encoded protein is an integral membrane protein that forms a voltage-independent calcium-activated channel with three other calmodulin-binding subunits.

Questions pertaining to this data can be directed to techsupport@avivasysbio.com

Aviva Systems Biology’s KCNN3 antibody (ARP35097_P050) has been tested using other cell lysates and tissues. To obtain more data about this antibody please email us at info@avivasysbio.com.

To order by phone call us at (888) 880-0001, fax us at (858) 552-6975 or send an email to info@avivasysbio.com. Aviva manufactures this antibody so we can offer the best price. Please contact us to request pricing information.

All of Aviva’s products are guaranteed for the applications and experimental sample types mentioned in the datasheet below. Are you curious if this product will work for you? Please contact us at techsupport@avivasysbio.com

Product Review:KCNN3 antibody-C-terminal region (ARP35097_P050) in Rhesus macaque spinal cord using IHC

Product Page for KCNN3 antibody-C-terminal region (ARP35097_P050)

Researcher:Timur Mavlyutov, University of Wisconsin Medical School
Application: IHC
Species+tissue/cell type:Rhesus macaque spinal cord
Primary antibody dilution: 1:300
Secondary antibody: Donkey anti Rabbit 488
Secondary antibody dilution: 1:500

Questionnaire:
1.Fixation method
-Perfusion by 4%PFA
2.Antigen retrieval method used
- permeabilization by 0.1% Triton X100
3.Primary antibody dilution
- All 1/300
4.Secondary antibody
-Donkey anti Rabbit 488
5.Secondary antibody dilution
-1/500

 
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