KCNN3 antibody - C-terminal region (ARP35097_P050)

Catalog#: ARP35097_P050

• Size: 50ug

Human Daudi

Mouse kidney, brain

Rhesus macaque spinal cord

• 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
• Alias Symbols: SK3; hSK3; SKCA3; KCa2.3
• Protein Accession#: NP_002240
• Nucleotide Accession#: NM_002249
• Swissprot Id: Q9UGI6
• Host: Rabbit
• Clonality: Polyclonal
• Protein Size (# AA): 736
• Molecular Weight: 81kDa
• Application: 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
• Predicted Homology Based on Immunogen Sequence: Bovine: 100%; Dog: 100%; Guinea pig: 100%; Human: 100%; Mouse: 100%; Pig: 100%; Rabbit: 100%; Rat: 100%
• Peptide Sequence: Synthetic peptide located within the following region: ITELNDRSEDLEKQIGSLESKLEHLTASFNSLPLLIADTLRQQQQQLLSA
• Blocking Peptide: For anti-KCNN3 antibody is Catalog # AAP35097 (Previous Catalog # AAPP06328)
• Key Reference: Kolski-Andreaco,A., et al., (2004) J. Biol. Chem. 279 (8), 6893-6904
• Reconstitution and Storage: Add 50 μl of distilled water. Final anti-KCNN3 antibody concentration is 1 mg/ml in PBS buffer. For longer periods of storage, store at -20°C. 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.

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